Use of the protein maba (fabg1) of mycobacterium tuberculosis for designing and screening antibiotics

ABSTRACT

The invention especially relates to the protein MabA, also named protein FabG1, which is recombinant in a purified form, or the recombinant proteins derived from the protein MabA by mutation of at least one amino acid. The invention also relates to the uses of said proteins and to the crystallographic co-ordinates thereof, in terms of the implementation of methods for designing and screening ligands of said proteins, and advantageously, ligands inhibiting the enzymatic activity of said proteins.

The main subject of the present invention is the use of the proteinMabA, and derived proteins, and more particularly the crystallographicco-ordinates of these proteins, within the framework of theimplementation of methods for designing and screening ligands of theseproteins, and advantageously ligands inhibiting the enzymatic activityof these proteins, namely antibiotics capable of being used within theframework of the treatment of mycobacteriosis.

Tuberculosis is one of the major causes of mortality by a singleinfectious agent, Mycobacterium tuberculosis. Moreover, for aboutfifteen years, there has been a recrudescence of this disease inindustrialized countries. This phenomenon is linked in part to theappearance of antiobiotic-resistent strains of Mycobacteriumtuberculosis. Thus, the design of new antituberculous-medicaments hasbecome a declared priority of the Word Health Organization.

The targets of the antituberculous antibiotics currently used in clinicsform-part of biosynthesis metabolisms of components of the envelope ofMycobacterium tuberculosis. In particular, the target of isoniazid(INH), a 1st-line antituberculous agent, is involved in the synthesis ofvery long-chain fatty acids (C60-C90), the mycolic acids. Isoniazidinhibits the activity of the protein InhA, which forms part of an enzymecomplex, FAS-II, the function of which is to produce, by successiveelongation cycles, long-chain fatty acids (C18-C32), precursors of themycolic acids. InhA, a 2-trans-enoyl-ACP reductase, catalyzes the 4thstage of an elongation cycle, which comprises 4 stages. INH is apro-drug which forms, with the coenzyme of InhA, NADH, an inhibitingadduct, INI-NAD(H). FAS-II comprises at least 3 other main enzymes, thelatter therefore representing potential targets for novel antibiotics.The protein MabA catalyzes the 2nd stage of the cycle.

The present invention provides methods for the design of antibiotics forthe treatment of mycobacterial infections, in particular tuberculosis.This invention deals with the mabA (fabG1, Rv1483) gene of Mycobacteriumtuberculosis, the product of this gene, the protein MabA (FabG1), aswell as with the material and methods used for the production of theprotein in a large quantity, the determination of its three-dimensionalstructure on the atomic scale and the study of its interactions withdifferent ligands or their effect on its enzymatic activity. Theinvention is based on the use of the protein MabA as a target forantibiotics; in particular, the study of the interaction of MabA withdifferent ligands or their effect on its enzymatic activity, bydifferent methods, is used in order to design inhibitors of theenzymatic activity of MabA.

The-present invention provides the methodological tools and materialnecessary for designing molecules representing potentialanti-mycobacterial and antituberculous antibiotics.

The present invention proposes the biological material and themethodologies necessary for the production and purification, in largequantities, of a potential target of antituberculous antibiotics, theprotein MabA. Moreover, these stages can be carried out very rapidlythanks to the overproduction of MabA provided with a poly-Histidine tagin Escherichia coli and its purification in a single stage by metalchelation chromatography, producing a protein with a high degree ofpurity. The quantity and quality of the purified protein make itpossible to obtain reliable results during studies of enzymatic activityor binding of ligands, but also allow the crystallization of the proteinin order to resolve its three-dimensional structure. The development ofconditions allowing the freezing of the MabA crystals in liquid nitrogenhas made it possible to obtain sets of atomic resolution data (2.05 Åcompared with 2.6 Å at ambient temperature) and opens the way to betterdata thanks to the use of synchrotron radiation. The frozen crystallinestructure has revealed the role of compounds (in particular caesium)necessary for the growth of the MabA crystals and makes it possible toenvisage rational optimization of crystal growth. The screening incrystallo of “pools” of ligands can also be carried out. The quantity ofprotein purified is also an important criterion for carrying out highthrough-put screenings of combinatorial libraries (see below).

The protein MabA activity tests, and as a result the tests on inhibitionby potential inhibitors, can be followed easily and rapidly byspectrophotometry, by monitoring the oxidizing of the reductioncoenzyme, NADPH, at 340 nm. The inhibition constants (IC50 and Ki) andthe inhibition mechanism (competitive, non-competitive, uncompetitiveinhibition) for each molecule can be deduced from this. Moreover, testson specific binding of ligands to the active site of MabA can be alsocarried out easily and rapidly, by spectrofluorimetry. The presence ofthe single Trp residue of the protein in the active site makes itpossible, by excitation at 303 nm, to detect, from the variation in thefluorescence emission intensity at the emission maximum, the binding ofa ligand and to deduce from this the disassociation constant (Kd).Similarly, FRET (Fluorescence Resonance Energy Transfer) experiments canbe carried out in the presence of the coenzyme, NADPH, making itpossible to conclude from this whether or not the ligand occupies thebinding site of the NADPH (binding competition). The simplicity of thesemeasurement methods, and the relatively low volumes that they require,will allow a miniaturization of the inhibition or ligand-binding tests,for the automatic high through-put screening of combinatorial libraries,thanks to an automatic device provided with a spectrophotometer orspectrofluorimeter.

Comparison of the structure of MabA with that of the-protein InhA, aprotein of the same structural super-family (RED) and belonging to thesame enzyme complex (see above), suggested an inhibiting effect ofisoniazid on MabA activity and detection of the active form of isoniazid(antituberculous), the INH-NADP(H) adduct. The binding of the adduct andinhibition of the MabA activity was then confirmed experimentally.Similarly, thanks to a strong structural similarity with other proteins,which have been or will be crystallized with ligands (for example,steroid derivatives, co-crystallized with steroid dehydrogenases), therational design of potential MabA inhibitors can be carried out rapidly.

The protein MabA is of particular interest as a target ofanti-mycobacterial antibiotics. In fact, it forms part of the sameenzymatic system as the protein InhA, target of the 1st-lineantituberculous medicament, isoniazid. On the other hand, up to now, noprotein homologous to MabA has been detected in animal cells. Moreover,comparison with the homologous proteins found in bacteria or plants hashighlighted particular properties of MabA, which are linked to itsfunction, since it uses long chain substrates. These characteristics arereflected in the structure of its active site, which makes it possibleto envisage the design of inhibitors specific to MabA (in particular, interms of size and hydrophobic character), and therefore ofnarrow-spectrum antibiotics. These different points provide MabA withcriteria for pharmacological credibility.

Thus, the main object of the present invention is:

research into and design of medicaments effective against opportunistmycobacterial infections (M. avium, M. kansasii, M. fortuitum, M.chelonae etc.) presenting problems in hospitals (sterilization ofmedical instruments), and in the case of human immuno-deficiency (AIDS,administration of immunosuppressors during a graft, in the case ofcancers etc.).

research into and design of medicaments effective against tuberculousinfections, in particular medicaments which are effective on the strainsof M. tuberculosis resistant to the antibiotics currently used inantituberculous therapy, and which are propagated in populations at risk(prison environment, economically disadvantaged environments etc.).

research into and design of medicaments effective against otherbacterial infections, by taking proteins homologous to MabA as-moleculartargets.

A main subject of the present invention is the protein MabA, also calledprotein FabG1, recombinant in-the purified form, or the recombinantproteins derived from the protein MabA by mutation of one or more aminoacids, said derived proteins being in purified form, and having anNADPH-dependent β-ketoacyl reductase activity.

A more particular subject of the invention is the purified recombinantprotein MabA, said protein being a protein of mycobacteria, such asMycobacterium tuberculosis, and more particularly M. tuberculosis strainH37Rv.

A subject of the invention is also the recombinant protein MabA or theabovementioned derived recombinant proteins, in purified form, asobtained by transformation of strains of E. coli with a plasmidcontaining a sequence comprising the gene coding for the protein MabA,or comprising a sequence coding for a protein derived from MabA asdefined above, followed by a purification stage during which:

the abovementioned recombinant E. coli bacteria are washed in a washingbuffer, then taken up in a lysis buffer, and lysed by a freeze/thawcycle in the presence of protease inhibitors and lysozyme,

after treatment by DNAse I and RNAse A, in the presence of MgCl₂, andcentrifugation, the lysis supernatant of the bacteria obtained in thepreceding stage, to which 10% (v/v) of glycerol, or 400 μM of NADP⁺ isadded, is applied to an Ni-NTA agarose resin column,

after several washings with 5 mM buffer then 50 mM imidazole, theprotein MabA, or the derived protein, is eluted with the elution buffer.

According to an embodiment of the invention, the recombinant proteinMabA or the abovementioned derived recombinant proteins, in purifiedform, are obtained according to the process described above in which thedifferent bacteria washing, lysis, washing, and elution buffers are thefollowing:

bacteria washing buffer: 10 mM potassium phosphate, pH 7.8,

lysis buffer: 50 mM potassium phosphate, pH 7.8 containing 500 mM ofNaCl, 5 mM of imidazole,

washing buffer: 50 mM potassium phosphate, pH 7.8 containing 500 mM ofNaCl, 5 and 50 mM of imidazole,

elution buffer: 50 mM potassium phosphate, pH 7.8 containing 500 mM ofNaCl, and 175 mM of imidazole.

Advantageously, the proteins obtained using the :abovementioned buffersare used within the framework of enzymatic kinetic studies for thescreening of ligands according to the methods described hereafter.

According to another embodiment of the invention, the recombinantprotein MabA or the abovementioned derived recombinant proteins, inpurified form, are obtained according to the process described above inwhich the different bacteria washing, lysis, washing, and elutionbuffers are the following:

bacteria washing buffer: Tris 10mM, pH 8.0,

lysis buffer:

-   -   50 mM Tris buffer, pH 8.0, supplemented with 300 mM LiSO₄ and 5        mM imidazole;    -   or 50 mM Tris buffer, pH 8.0, supplemented with 300 mM KCl and 5        mM imidazole,

washing buffer:

-   -   50 mM Tris buffer, pH 8.0, supplemented with 300 mM LiSO4 and 5        or 50 mM imidazole,    -   or 50 mM Tris buffer, pH 8.0, supplemented with 300 mM KCl and 5        or 50 mM imidazole.

elution buffer:

-   -   20 mM MES buffer, pH 6.4, LiSO4 300 mM and 175-750 mM imidazole;    -   or 20 mM PIPES buffer, pH 8.0, supplemented with 300 mM KCl and        175-750 mM imidazole,

1 mM DTT being added to these buffers in the case of the wild-typeprotein MabA.

Advantageously, the proteins obtained using the abovementioned buffersare used within the framework of crystallography studies for designingand screening ligands according to the methods described hereafter.

The invention also relates to the abovementioned proteins derived fromthe abovementioned protein MabA, and characterized in that theycorrespond to the protein MabA the amino acid sequence SEQ ID NO: 1 ofwhich is the following: MTATATEGAK PPFVSRSVLV TGGNRGIGLA IAQRLAADGHKVAVTHRGSG APKGLFGVEC DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRMTEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGSWGIGNQ ANYAASKAGVIGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGVVSFLASEDAS YISGAVIPVD GGMGMGH

in which the cysteine in position 60 is replaced by a valine residue,and/or the glycine in position 139 is replaced by an alanine or aserine, and/or the serine in position 144 is replaced by a leucineresidue.

A more particular subject of the invention is therefore the proteinderived from the protein MabA as defined above, and characterized inthat it corresponds to the protein MabA in which the cysteine inposition 60 is replaced by a valine residue, said derived protein, alsocalled C(60)V, corresponding to the following sequence SEQ ID NO 3:MTATATEGAK PPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEVDVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVAQRASRSMQRN KFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANVVAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVDGGMGMGH

A more particular subject of the invention is therefore also the proteinderived from the protein MabA as defined above, and characterized inthat it corresponds to the protein MabA in which the serine in position144 is replaced by a leucine residue, said derived protein, also calledS(144)L, corresponding to the following sequence SEQ ID NO 5: MTATATEGAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

A more particular subject of the invention is therefore also the proteinderived from the protein MabA as defined above, and characterized inthat it corresponds to the protein MabA in which the cysteine inposition 60 is replaced by a valine residue, and the serine in position144 is replaced by a leucine residue, said derived protein, also calledC(60)V/S(144)L, corresponding to the following sequence SEQ ID NO 7:MTATATEGAK PPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEVDVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVAQRASRSMQRN KFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANVVAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVDGGMGMGH

A more particular subject of the invention is also the protein derivedfrom the protein MabA as defined above, and characterized in that itcorresponds to the protein MabA in which the cysteine in position 60 isreplaced by a valine residue, the glycine in position 139 is replaced byan alanine or a serine, and the serine in position 144 is replaced by aleucine residue, said derived protein, also called C(60)V/G(139)[A orS]/S(144)L, corresponding-to the following sequence SEQ ID NO 8:MTATATEGAK PPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEVDVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVAQRASRSMQRN KFGRMIFIXS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANVVAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVDGGMGMGH

in which X represents A or S.

The invention also relates to the protein MabA corresponding to thesequence SEQ ID NO: 1, or the proteins derived from the protein MabAdefined above, such as the derived proteins corresponding to thesequences SEQ ID NO: 3, 5, 7, or 8, characterized in that they aremodified such that they include one or more mutations making it possibleto change the specificity of the protein NADPH to NADH.

A more particular subject of the invention is the abovementionedmodified proteins MabA, corresponding to the following sequences:

the sequence SEQ ID NO: 9, corresponding to the sequence SEQ ID NO: 1comprising the mutations N24D(or E), and/or H46D, namely the followingsequence: MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSGAPKGLFGVEC DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVINANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARELSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDASYISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the sequence SEQ ID NO: 10, corresponding to the sequence SEQ ID NO: 3comprising the mutations N24D(or E), and/or H46D, namely the followingsequence: MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSGAPKGLFGVEV DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVINANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARELSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDASYISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the sequence SEQ ID NO: 11, corresponding to the sequence SEQ ID NO: 5comprising the mutations N24D(or E), and/or H46D, namely the followingsequence: MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSGAPKGLFGVEC DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVINANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARELSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDASYISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the sequence SEQ ID NO: 12, corresponding to the sequence SEQ ID NO: 7comprising the mutations N24D(or E), and/or H46D, namely the followingsequence: MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSGAPKGLFGVEV DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVINANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARELSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDASYISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the sequence SEQ ID NO: 13, corresponding to the sequence SEQ ID NO: 8comprising the mutations N24D(or E), and/or H46D, namely the followingsequence: MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSGAPKGLFGVEV DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVINANLTGAFRVA QRASRSMQRN KFGRMIFIXS VSGSWGIGNQ ANYAASKAGV IGMARSIARELSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDASYISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D.

A subject of the invention is also the protein MabA corresponding to thesequence SEQ ID NO: 1, or the proteins derived from the protein MabAdefined above, such as the derived proteins corresponding to thesequences SEQ ID NO: 3, 5, 7, 8, 9, 10, 11, 12, or 13, characterized inthat they are modified by insertion, on the N-terminal side, of apoly-histidine tag such as the following sequence SEQ ID NO: 14:MGSSHHHHHH SSGLVPRGSH.

A more particular subject of the invention is the abovementionedmodified proteins MabA, corresponding to the following sequences:

the sequence SEQ ID NO: 15, corresponding to the combination of thesequence SEQ ID NO: 14 and the sequence SEQ ID NO: 1, namely thefollowing sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLVTGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEC DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the sequence SEQ ID NO: 16, corresponding to the combination of thesequence SEQ ID NO: 14 and the sequence SEQ ID NO: 3, namely thefollowing sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLVTGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the sequence SEQ ID NO: 17, corresponding to the combination of thesequence SEQ ID NO: 14 and the sequence SEQ ID NO: 5, namely thefollowing sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLVTGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEC DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the sequence SEQ ID NO: 18, corresponding to the combination of thesequence SEQ ID NO: 14 and the sequence SEQ ID NO: 7, namely thefollowing sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLVTGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the sequence SEQ ID NO: 19, corresponding to the combination of thesequence SEQ ID NO: 14 and the sequence SEQ ID NO: 9, namely thefollowing sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLVTGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEC DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the sequence SEQ ID NO: 20, corresponding to the combination of thesequence SEQ ID NO: 14 and the sequence SEQ ID NO: 10, namely thefollowing sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLVTGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the sequence SEQ ID NO: 21, corresponding to the combination of thesequence SEQ ID NO: 14 and the sequence SEQ ID NO: 11, namely thefollowing sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLVTGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEC DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the sequence SEQ ID NO: 22, corresponding to the combination of thesequence SEQ ID NO: 14 and the sequence SEQ ID NO: 12, namely thefollowing sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLVTGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the sequence SEQ ID NO: 23, corresponding to the combination of thesequence SEQ ID NO: 14 and the sequence SEQ ID NO: 13, namely thefollowing sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLVTGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIXSVSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D.

A subject of the invention is also the protein MabA corresponding to thesequence SEQ ID NO: 1, or the proteins derived from the protein MabAdefined above, such as the derived proteins corresponding to thesequences SEQ ID NO: 3, 5, 7, 8, 9, 10, 11, 12, or 13, having anN-terminal GSH sequence, namely the following sequences:

the following sequence SEQ ID NO: 24, corresponding to the combinationof the GSH sequence and the sequence SEQ ID NO: 1, GSH MTATATEGAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 25, corresponding to the combinationof the GSH sequence and the sequence SEQ ID NO: 3, GSH MTATATEGAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 26, corresponding to the combinationof the GSH sequence and the sequence SEQ ID NO: 5, GSH MTATATEGAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 27, corresponding to the combinationof the GSH sequence and the sequence SEQ ID NO: 7, GSH MTATATEGAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 28, corresponding to the combinationof the GSH sequence and the sequence SEQ ID NO: 9, GSH MTATATEGAKPPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the following sequence SEQ ID NO: 29, corresponding to the combinationof the GSH sequence and the sequence SEQ ID NO: 10, GSH MTATATEGAKPPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the following sequence SEQ ID NO: 30, corresponding to the combinationof the GSH sequence and the sequence SEQ ID NO: 11, GSH MTATATEGAKPPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the following sequence SEQ ID NO: 31, corresponding to the combinationof the GSH sequence and the sequence SEQ ID NO: 12, GSH MTATATEGAKPPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the following sequence SEQ ID NO: 32, corresponding to the combinationof the GSH sequence and the sequence SEQ ID NO: 13, GSH MTATATEGAKPPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D.

A subject of the invention is also the protein MabA corresponding to thesequence SEQ ID NO: 1, or the proteins derived from the protein MabAdefined above, such as the derived proteins corresponding to thesequences SEQ ID NO: 3, 5, 7, 8, 9, 10, 11, 12, or 13, the first sevenamino acids of which are deleted, namely the following sequences:

the following sequence SEQ ID NO: 33, corresponding to the sequence SEQID NO: 1 the first seven amino acids of which are deleted: GAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 34, corresponding to the sequence SEQID NO: 3 the first seven amino acids of which are deleted: GAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 35, corresponding to the sequence SEQID NO: 5 the first seven amino acids of which are deleted: GAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 36, corresponding to the sequence SEQID NO: 7 the first seven amino acids of which are deleted: GAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 37, corresponding to the sequence SEQID NO: 9 the first seven amino acids of which are deleted: GAKPPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the following sequence SEQ ID NO: 38, corresponding to the sequence SEQID NO: 10 the first seven amino acids of which are deleted: GAKPPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the following sequence SEQ ID NO: 39, corresponding to the sequence SEQID NO: 11 the first seven amino acids of which are deleted: GAKPPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the following sequence SEQ ID NO: 40, corresponding to the sequence SEQID NO: 12 the first seven amino acids of which are deleted: GAKPPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D,

the following sequence SEQ ID NO: 41, corresponding to the sequence SEQID NO: 13 the first seven amino acids of which are deleted: GAKPPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D.

The invention also relates to the protein MabA and the abovementionedderived proteins, characterized by their specific enzymatic activity ofthe substrates of the long-chain type β-ketoacyl, in particular between8 and 20 carbon atoms, such as β-ketooctanoyl-CoA, orβ-ketododecanoyl-CoA.

A more particular subject of the invention is the protein MabA and theabovementioned derived proteins, the main characteristics of thethree-dimensional structure of which, at a resolution of 1.6-2.0angströms, detected by X-ray diffraction analysis of the crystals ofsaid proteins, are as represented in FIG. 1 for the recombinant proteinMabA corresponding to the sequence SEQ ID NO: 15, in FIG. 2 for thederived protein MabA C(60)V corresponding to the sequence SEQ ID NO: 16,and in FIG. 3 for the derived protein MabA C(60)V/S(144)L correspondingto the sequence SEQ ID NO: 17.

The invention also relates to the protein MabA and the abovementionedderived proteins, in crystallized form.

The invention relates more particularly to the crystals ofabovementioned proteins, as obtained by the hanging-drop vapourdiffusion method, by mixing said proteins (1 μl of a 10 mg/ml solution)with a solution of polyethylene glycol, CsCl (150-300 mM), and glycerol(10%) in a buffer (PIPES) at pH 6.2.

A subject of the invention is also the crystals of abovementionedproteins, as obtained according to the crystallization method describedabove, said method being carried out from proteins purified using theabovementioned buffers more particularly used for obtaining proteins ofthe invention intended for crystallography studies.

The invention also relates to the abovementioned crystals of therecombinant protein MabA corresponding to the sequence SEQ ID NO: 15,the atomic coordinates of the three-dimensional structure of which arerepresented in FIG. 1, and having the following characteristics:

cell parameters:

-   -   a=81.403 angströms, b=116.801 angströms, c=52.324 angströms,    -   α=β=90.00°, γ=122.30°,

space group: C2,

maximum diffraction=2.05 angströms.

The invention also relates to the abovementioned crystals of the proteinC(60)V corresponding to the sequence SEQ ID NO: 16, the atomiccoordinates of the three-dimensional structure of which are representedin FIG. 2, and having the following characteristics:

cell parameters:

-   -   a=82.230 angströms, b=118.610 angströms, c=53.170 angströms,    -   α=β=90.00°, γ=122.74°,

space group: C2,

maximum diffraction=2.6 angströms.

A subject of the invention is also the abovementioned crystals of theprotein C(60)V/S(144)L corresponding to the sequence SEQ ID NO: 18, theatomic coordinates of the three-dimensional structure of which arerepresented in FIG. 3, and having the following characteristics:

cell parameters:

-   -   a=81.072 angströms, b=117.022 angströms, c=53.170 angströms,    -   α=β=90.00°, γ=122.42°,

space group: C2,

maximum diffraction=1.75 angströms.

A more particular subject of the invention is the crystals of MabA andabovementioned derived proteins, in which said proteins are bound to aligand, namely a molecule capable of binding to the protein MabA or tothe proteins derived from the latter, more particularly at the level oftheir active site mainly delimited by the amino acids situated inpositions 21 to 28, 45 to 48, 60 to 63, 87 to 100, 112, 138 to 157, 183to 212, and 240 to 247 of the proteins corresponding to the sequencesSEQ ID NO: 1, 3, 5, 7, 8, 9, 10, 11, or 13, or in positions 41 to 48, 65to 68, 80 to 83, 107 to 120, 132, 158 to 177, 203 to 232, and 260 to267, of the proteins corresponding to the sequences SEQ ID NO: 15 to 23,or in positions 24 to 31, 48 to 51, 63 to 66, 90 to 103, 115, 141 to160, 186 to 215, and 243 to 250, of the proteins corresponding to thesequences SEQ ID NO: 24 to 32, or in positions 14 to 11, 38 to 41, 53 to56, 80 to 93, 105, 131 to 150, 176 to 205, and 233 to 240, of theproteins corresponding to the sequences SEQ ID NO: 33 to 41, saidcrystals being as obtained by soaking or co-crystallization of therecombinant protein MabA in purified form, or of a recombinant proteinderived from the abovementioned protein MabA, in the presence of saidligand, in particular under the crystallization conditions definedabove.

The invention also relates to the nucleotide sequences coding for aprotein derived from the protein MabA as defined above.

A more particular subject of the invention is therefore the nucleotidesequence coding for the derived protein C(60)V (SEQ ID NO: 3), andcorresponding to the following sequence SEQ ID NO: 2:atgactgccacagccactgaaggggccaaacccccattcgtatcccgttcagtcctggttaccggaggaaaccgggggatcgggctggcgatcgcacagcggctggctgccgacggccacaaggtggccgtcacccaccgtggatccggagcgccaaaggggctgtttggcgtcgaagttgacgtcaccgacagcgacgccgtcgatcgcgccttcacggcggtagaagagcaccagggtccggtcgaggtgctggtgtccaacgccggcctatccgcggacgcattcctcatgcggatgaccgaggaaaagttcgagaaggtcatcaacgccaacctcaccggggcgttccgggtggctcaacgggcatcgcgcagcatgcagcgcaacaaaattcggtcgaatgatattcataggttcggtctccggcagctggggcatcggcaaccaggccaactacgcagcctccaaggccggagtgattggcatggcccgctcgatcgcccgcgagctgtcgaaggcaaacgtgaccgcgaatgtggtggccccgggctacatcgacaccgatatgacccgcgcgctggatgagcggattcagcagggggcgctgcaatttatcccagcgaagcgggtcggcacccccgccgaggtcgccggggtggtcagcttcctggcttccgaggatgcgagctatatctccggtgcggtcatcccggtcgacggcggcatgggtatgggccac

or any sequence derived by degeneration of the genetic code and codingfor the protein C(60)V.

A subject of the invention is therefore also the nucleotide sequencecoding for the derived protein S(144)L (SEQ ID NO: 5), and correspondingto the following sequence SEQ ID NO: 4:atgactgccacagccactgaaggggccaaacccccattcgtatcccgttcagtcctggttaccggaggaaaccgggggatcgggctggcgatcgcacagcggctggctgccgacggccacaaggtggccgtcacccaccgtggatccggagcgccaaaggggctgtttggcgtcgaatgtgacgtcaccgacagcgacgccgtcgatcgcgccttcacggcggtagaagagcaccagggtccggtcgaggtgctggtgtccaacgccggcctatccgcggacgcattcctcatgcggatgaccgaggaaaagttcgagaaggtcatcaacgccaacctcaccggggcgttccgggtggctcaacgggcatcgcgcagcatgcagcgcaacaaaattcggtcgaatgatattcataggttcggtctccggcctctggggcatcggcaaccaggccaactacgcagcctccaaggccggagtgattggcatggcccgctcgatcgcccgcgagctgtcgaaggcaaacgtgaccgcgaatgtggtggccccgggctacatcgacaccgatatgacccgcgcgctggatgagcggattcagcagggggcgctgcaatttatcccagcgaagcgggtcggcacccccgccgaggtcgccggggtggtcagcttcctggcttccgaggatgcgagctatatctccggtgcggtcatcccggtcgacggcggcatgggtatgggccac

or any sequence derived by degeneration of the genetic code and codingfor the protein S(144)L.

A subject of the invention is therefore also the nucleotide sequencecoding for the derived protein C(60)V/S(144)L (SEQ ID NO: 7), andcorresponding to the following sequence SEQ ID NO: 6:atgactgccacagccactgaaggggccaaacccccattcgtatcccgttcagtcctggttaccggaggaaaccgggggatcgggctggcgatcgcacagcggctggctgccgacggccacaaggtggccgtcacccaccgtggatccggagcgccaaaggggctgtttggcgtcgaagttgacgtcaccgacagcgacgccgtcgatcgcgccttcacggcggtagaagagcaccagggtccggtcgaggtgctggtgtccaacgccggcctatccgcggacgcattcctcatgcggatgaccgaggaaaagttcgagaaggtcatcaacgccaacctcaccggggcgttccgggtggctcaacgggcatcgcgcagcatgcagcgcaacaaaattcggtcgaatgatattcataggttcggtctccggcctctggggcatcggcaaccaggccaactacgcagcctccaaggccggagtgattggcatggcccgctcgatcgcccgcgagctgtcgaaggcaaacgtgaccgcgaatgtggtggccccgggctacatcgacaccgatatgacccgcgcgctggatgagcggattcagcagggggcgctgcaatttatcccagcgaagcgggtcggcacccccgccgaggtcgccggggtggtcagcttcctggcttccgaggatgcgagctatatctccggtgcggtcatcccggtcgacggcggcatgggtatgggccac

or any sequence derived by degeneration of the genetic code and codingfor the protein C(60)V/S(144)L.

The invention also relates to any recombinant nucleotide sequencecomprising the nucleotide sequence coding for the protein MabA, orcomprising a nucleotide sequence coding for a protein derived from theprotein MabA, as defined above, in combination with the elementsnecessary for the transcription of this sequence, in particular with atranscription promoter and terminator.

A subject of the invention is also any vector, in particular plasmid,containing a nucleotide sequence as defined above.

The invention also relates to the host cells transformed by anabovementioned vector, said cells being chosen in particular frombacteria such as E. coli, or any other microorganism used for theproduction of proteins.

A subject of the invention is also a process for the preparation of therecombinant protein MabA in purified form, or of recombinant proteinsderived from the protein MabA, as defined above, characterized in thatit comprises the following stages:

transformation of cells using an abovementioned recombinant vector,

culture of the cells thus transformed, and recovery of said proteinsproduced by said cells,

purification of said proteins according to the purification processdescribed above.

The invention also relates to the use of the recombinant protein MabA inpurified form, or of recombinant proteins derived from the protein MabAas defined above, or of abovementioned crystals, for the implementationof methods for designing or screening ligands of the protein MabA, andmore particularly molecules capable of binding specifically at the levelof the active site of the protein MabA, or proteins similar in structureto the protein MabA, and inhibiting the enzymatic activity of thelatter, these inhibitors being chosen in particular from:

the steroid derivatives,

the derivatives of the antituberculous antibiotic isoniazid(isonicotinic acid hydrazide), such as the derivatives of theisonicotinoyl-NAD(P) adduct,

the derivatives of N-acetyl cysteamine or other simplified types ofderivatives of the coenzyme A, comprising a grafted fluorophore makingit possible to use the fluorescence spectroscopy method, in particulartime-resolved, for the detection of protein-ligand interactions,

the inhibiting derivatives of the protein hihA of Mycobacteriumtuberculosis.

A more particular subject of the invention is the abovementioned use ofthe recombinant protein MabA in purified form, or recombinant proteinsderived from the protein MabA as defined above, or abovementionedcrystals, for the implementation of methods for designing or screeningligands of the protein MabA capable of being used in pharmaceuticalcompositions, in particular within the framework of the treatment ofpathologies linked to mycobacterial infections, such as tuberculosislinked to infection by Mycobacterium tuberculosis, or by Mycobacteriumafricanium, or leprosy linked to infection by Mycobacterium leprae, ormycobacteriosis linked to infection by opportunist mycobacteria, such asMycobacterium avium, Mycobacterium fortuitum, Mycobacterium kansasii,Mycobacterium chelonae.

The invention also relates to any method for screening ligands of theprotein MabA, characterized in that it comprises the following stages:

being brought into the presence of the recombinant protein MabA inpurified form, or a recombinant protein derived from the protein MabA asdefined above,

detection of any bond between said protein and the ligand tested bymeasurement, after fluorescence excitation, in particular at 300 nm, ofthe intensity of fluorescence of said protein emitted between 300 and400 nm (corresponding essentially to the emission of fluorescence of thesingle tryptophan W145), and comparison of the intensity of fluorescendeemitted in a test in the absence of ligand, the binding of a ligand inthe MabA active site being characterized by a quenching of fluorescence.

A subject of the invention is also any method for screening ligandsinhibiting the protein MabA, characterized in that it comprises thefollowing stages:

being brought into the presence of the recombinant protein MabA inpurified form, or a recombinant protein derived from the protein MabA asdefined above, in a reaction medium comprising a substrate, such as aβ-ketoacyl derivative defined above, the coenzyme NADPH and the ligandtested,

detection of a potential inhibiting ability of the ligand tested, bymeasurement of the enzymatic activity of said protein by kineticmeasurement of the absorbance, in particular at 340 nm, and comparisonof the gradient of the optical density curve as a function of time withthe gradient obtained in a test in the absence of ligand.

The invention also relates to any method for screening ligands of theprotein MabA, characterized in that it comprises the following stages:

being brought into the presence of the recombinant protein MabA inpurified form, or of a recombinant protein derived from the protein MabAas defined above, with the ligand tested,

analysis of the three-dimensional structure of the complex formed insoluble phase between said protein and said ligand, in particular byNMR, and by fluorescence.

A more particular subject of the invention is any method for screeningligands of the protein MabA, characterized in that it comprises thefollowing stages:

co-crystallization of the ligand tested and the recombinant protein MabAin purified form, or of a recombinant protein derived from the proteinMabA as defined above, in particular under the crystallizationconditions defined above, in order to obtain the abovementionedcrystals,

or soaking of the crystals of the protein MabA or of a derived proteinas defined above, in optimized solutions containing potential ligands,

analysis of the three-dimensional structure of the abovementionedcrystals, in particular by X-ray diffraction (with a view to selectingthe ligands having an optimum ability to occupy and block the activesite of said proteins).

The invention also relates to the use of the coordinates of thethree-dimensional structure of the recombinant protein MabA in purifiedform, or a recombinant protein derived from the protein MabA as definedabove, said coordinates being represented in FIGS. 1 to 3, ifappropriate in combination with the coordinates of the active site ofthese proteins as defined above, for the implementation of methods fordesigning or screening ligands of the protein MabA (advantageouslycomputer-aided).

A more particular subject of the invention is therefore any method fordesigning or screening ligands of the protein MabA, comprising the useof the coordinates of the three-dimensional structure of the recombinantprotein MabA in purified form, or of a recombinant protein derived fromthe protein MabA as defined above, said coordinates being represented inFIGS. 1 to 3, for screening in silico the virtual combinatoriallibraries of potential ligands, advantageously using appropriatecomputer software, and the detection and rational structuraloptimization of the molecules capable of binding to said protein.

A subject of the invention is also any method of rational design asdefined above, carried out starting with known inhibitors of MabA orinhibitors of proteins homologous to MabA (of the same SDR or REDstructural family, and exhibiting more than 10% identity with MabAthroughout the peptide sequence), for which the fine three-dimensionalstructure of the complex between said inhibitor and the recombinantprotein MabA in purified form, or a recombinant protein derived from theprotein MabA, as defined above, was determined, and rational structuraloptimization of said inhibitors. We have shown that the activity of MabAwas inhibited in vitro by an INH-NADP(H) adduct. This action mechanismof isoniazid (INH) on MabA is similar to the action mechanism ofisoniazid on the protein InhA, target of the INH. Other proteins formingpart of the RED superfamily have a three-dimensional structurecomparable to that of MabA, including a steroid dehydrogenase (PDB1HSD),tropinone reductases (e.g. PDB1AE1), a trihydroxynaphthalene reductase(PDB1YBV) and a mannitol dehydrogenase (PDB1H5Q), and wereco-crystallized with inhibitors.

The invention is further illustrated by means of the detaileddescription which follows of obtaining the recombinant protein MabA, andthe proteins MabA C(60)V, S(144)L, and C(60)V/S(144)L, in purified form,their enzymatic properties, as well as crystals of these proteins andtheir atomic coordinates.

The atomic coordinates of the recombinant protein MabA (corresponding toSEQ ID NO: 15), and the proteins MabA C(60)V(corresponding to SEQ ID NO:16), and C(60)V/S(144)L (corresponding to SEQ ID NO: 18), arerespectively represented in FIGS. 1 to 3, which show from left to rightthe atomic number, name of the residues, chain number, x, y, z,coordinates, occupation, and factor B.

EXPERIMENTAL PART

Tuberculosis, an infectious disease caused by Mycobacteriumtuberculosis, remains the major cause of mortality world-wide due to asingle infectious agent. According to the World Health Organization, 8million cases of tuberculosis appear each year, resulting in 3 milliondeaths (Dolin et al., 1994). Whilst it has always posed a serious publichealth problem in developing countries, tuberculosis is reappearing inthe developed countries. The precarious conditions of certain socialgroups and the deterioration in health systems, consequences of theworld economic crisis, have promoted this recrudescence of tuberculosis.Similarly, the endemic of infection by the human immunodeficiency virus(HIV) and the appearance of strains of M. tuberculosis resistant to oneor more antibiotics have also strongly contributed to this phenomenon(Barnes et al., 1991). The emergence of multi-resistant tuberculosis,defined as resistance to the two antibiotics which form the basis ofantituberculous treatment, isoniazid (Rimifon, INH) and rifampicin(RMP), is a threat to the control of tuberculosis. Patients infected bythe strains resistant to several antibiotics are extremely difficult tocare for and the necessary treatment is toxic and expensive. Up to 30%of all of the resistant clinical isolates of M. tuberculosis areresistant to isoniazid (Cohn et al., 1997). It is therefore important tobetter understand the mechanisms of resistance to isoniazid establishedby the mycobacteria in order to be able, on the one hand, to developrapid techniques allowing the detection of resistances and, on the otherhand, to develop new anti-mycobacterial agents which are effectiveagainst the resistant strains.

Starting from numerous works carried out on isoniazid, it has beenpossible to identify a direct target of this antibiotic: a metabolismspecific to mycobacteria, the biosynthesis of mycolic acids [(Winder &Collins, 1970); (Takayama et al., 1972); (Quémard et al., 1991)]. Thesevery long chain fatty acids are major and characteristic constituents ofthe mycobacterial envelope. Thanks to the use of the tools of molecularbiology in M. tuberculosis, a molecular target of isoniazid, the proteinInhA, probably involved in the biosynthesis route of mycolic acids hasbeen characterized [(Banejee et al., 1994); (Quemard et al., 1995)].InhA belongs to an enzymatic system responsible for the elongation ofthe fatty acids (Marrakchi et. al., 2000). This system containing theprotein InhA, a target of isoniazid, participates in the biosynthesis ofmycolic acids and therefore represents an enzymatic complex thecomponents of which are interesting to study, as potential targets ofnew antituberculous antibiotics. We have therefore studied thebiochemical properties of MabA, one of the proteins of the complexcontaining InhA. The molecular modelling of the three-dimensionalstructure of this protein, which catalyzes the reduction of β-ketoacylderivatives, has shown that MabA and InhA form part of the samestructural family. The study of the effect of isoniazid on the enzymaticactivity of MabA suggests that the antibiotic inhibits the protein by amechanism similar to the action on InhA. Thus, MabA represents a usefultarget for the design of inhibitors of the biosynthesis of fatty acidsin mycobacteria.

Within the framework of the work on the present invention, the mabA geneof M. tuberculosis was cloned in E. coli, in an expression vector. Theprotein is produced in a large quantity by this recombinant strain, as afusion protein possessing an N-terminal poly-histidine tag. Purificationof the protein is carried out in a single stage by columnchromatography, producing several mg of purified protein. A wild-typeMabA monomer possesses 247 amino acids and has a size of 25.7 kDa; thefusion monomer is 27.7 kDa. Several experimental methods (analyticalultracentrifugation, gel permeation, light diffusion, crystallographyetc.) made it possible for us to show that the native protein was mainlytetrameric, originating from the self-association of two dimers.Physico-chemical properties (stability in different buffer media, atdifferent temperatures, fluorescence emission spectra etc.) and the mainenzymatic properties of MabA (Kd, Km and kcat for the coenzyme and-β-ketoacyl-CoA- substrates of different chain lengths) were determined.The purified recombinant protein is functional; this is a β-ketoacylreductase, NADPH-dependent, and specific to long-chain substrates(C12-C20). We have shown that MabA formed part of the elongation systemof mycobacterial fatty acid, FAS-II, and catalyzes the 2nd stage of theelongation cycle.

The three-dimensional structure of the protein MabA was resolved bycrystallography to 2.05 Å resolution after development of the conditionsfor cryostabilization of the crystals. MabA forms part of the structuralsuper-family of the SDR (Short-Chain Reductases) or RED (Reductases,Epimerases, Dehydrogenases) proteins. It is homologous to the KARs(ketoacyl-ACP reductases), but represents a particular member of thisfamily, by of the structure of the substrate-binding pocket. The latterhas a more hydrophobic character than that of the homologous proteins.The presence of the single tryptophan residue in the substrate-bindingpocket allowed us to carry out fluorescence spectroscopy experiments,which demonstrated a more marked affinity of MabA for long-chainsubstrates (C8-C20) compared with the C4 substrate. These results, whichcorrelate with the enzymatic kinetic data, demonstrate astructure-function relation between the hydrophobicity of the bindingsite of the substrate and the affinity of MabA for unusually longsubstrates in the bacteria.

The distinct properties of MabA relative to the other homologousproteins make it a target of choice for the design of potentialantibiotics. This design will use several parallel approaches:

1. rational design starting with known inhibitors of MabA or homologousproteins

2. high-throughput screening of virtual combinatorial libraries

3. high-throughput screening of real combinatorial libraries.

We have shown that the activity of MabA was inhibited in vitro by anINH-NADP(H) adduct. This action mechanism of isoniazid (INH) on MabA issimilar to the action mechanism of isoniazid on the protein InhA, targetof NH. Other proteins forming part of the RED super-family have athree-dimensional structure comparable to that of MabA, including asteroid dehydrogenase (PDB1HSD), tropinone reductases (e.g.: PDB1AE1), atrihydroxynaphthalene reductase (PDB1YBV) and a mannitol dehydrogenase(PDB1H5Q). Thus, approach (1) is based on the use of the structure ofligands (e.g. isoniazid derivatives, steroids) of these differentproteins for the design of other potential inhibitors of MabA, ofderived structures. Rational design of course involves the use of thecrystalline structure of MabA and of the computer-aided moleculardocking method. Similarly, if approaches (2) and (3) provide new typesof potential ligands, the latter will be able to form the basis of newrational designs.

The invention therefore provides a conceptual approach for thedevelopment of inhibitors of the activity of the protein MabA. It alsooffers a method of experimental validation, on the one hand, of thespecific binding of these molecules to the active site of MabA(fluorescence spectroscopy) and on the other hand, of the inhibitingability of these molecules by a simple enzymatic test (enzymatickinetics by monitoring by spectrophotometry).

I) Study of the Protein MABA

We have shown that the FAS-II elongation system contains the proteinInhA, a target of isoniazid. Moreover, the fact that this system isprobably involved in the biosynthesis of mycdlic acids, compoundsspecific to mycobacteria, makes FAS-II a target of choice foranti-mycobacterial agents. Study of the enzymes which make up thissystem is therefore a useful approach for research into new targets ofantibiotics.

The strong inhibition of the activity of FAS-II by isoniazid, and aboveall the fact that no biosynthesis intermediate, and in particular thesubstrates of InhA, accumulate under the effect of the INH suggest thatanother target of the antibiotic could exist in addition to InhA. Theprotein MabA, coded by a gene contiguous to inhA on the chromosome of M.tuberculosis, probably forms part of the same enzymatic system as InhA.Several data suggest that MabA could be a target of isoniazid. On theone hand, point mutations in the promoter region of the mabA-inhA locusof clinical isolates of M. tuberculosis lead to the overproduction ofthe proteins downstream and correlate with a phenotype of resistance toINH. This suggests that in addition to the overproduction of InhA,induced by these mutations, the overproduction of MabA could alsoparticipate in the resistance, if this protein interacts with isoniazid.On the other hand, study of the effect of isoniazid (2 mM) on thepurified enzymes of the isolated FES system of M. avium has shown thattwo stages of the system are sensitive to INH, β-ketoacyl reductase (93%inhibition and Ki=353 μM), which is the most sensitive, andenoyl-reductase (26% inhibition and Ki=5.5 mM) (Kikuchi et al., 1989).

We therefore decided to purify the protein MabA and to study certain ofits biochemical properties, by adopting a strategy of overproduction ofthe protein in a prokaryotic system.

1. Overproduction of MABA in Escherichia Coli

Carrying out an enzymatic study and producing anti-MabA antibodies forthe intracellular location of MabA, required the obtaining of the pureprotein, in soluble form and in a large quantity. In order tooverproduce MabA, we used a system of expression and purification inEscherichia coli, which is simple and very effective for theoverexpression of prokaryotic genes. The development of an experimentalprocedure in order to achieve sufficient overproduction, whilstobtaining the protein in soluble form, required optimization at severallevels of the overexpression and purification diagram.

1.1 Cloning of the mabA Gene of Mycobacterium Tuberculosis H37Rv

The determination of the complete sequence of the genome ofMycobacterium tuberculosis H37Rv (Cole et al., 1998) and the developmentof the techniques of molecular biology allowing the manipulation ofrecombinant DNA, facilitated the production and study of the protein ofinterest, MabA.

1.1.1 Description of the Expression System of mabA in Escherichia Coli

Choice of the Expression Vector pET (Plasmid for Expression by T7 RNAPolymerase)

In the expression vector used, pET-15b (Novagen), the target gene iscloned under the control of the transcription and translation signals ofthe bacteriophage T7. The mabA (fabG1) gene of 741 base pairs, codingfor the protein MabA was amplified by polymerase chain reaction (PCR)from the cosmid MTCY277 (Institut Pasteur), and cloned between therestriction sites NdeI and Xho of the plasmid. This plasmid offers theadvantage of being able to obtain in NH2-terminal fusion of therecombinant protein, a poly-histidine sequence, cleavable, allowing arapid purification of the protein by affinity chromatography. Theconstruction therefore comprises upstream of the mabA gene, a sequencecoding for 6 successive histidines and the site of cleavage by thrombin.

Choice of the Host Strain

The host strain of E. coli chosen, BL21(λDE3) (Novagen), has theadvantage of having the 2 inactive ompT and lon genes. The ompT[(Studier & Moffatt, 1986); (Studier et al., 1990)] and lon genes(Phillips et al., 1984) code respectively for the parietal protease(responsible for the degradation of heterologous proteins) and the maincytoplasmic protease (responsible for the degradation of poorly foldedor unstable proteins). BL21(λDE3) is lysogen for the bacteriophage DE3(λ derivative), and therefore carries a chromosomal copy of the gene ofthe T7 RNA polymerase under the control of the lacUV5 promoter, which isIPTG (isopropyl-β-D-thiogalactopyranoside) inducible. The addition ofIPTG to a culture of the lysogen induces the expression of T7 RNApolymerase, which in turn will transcribe the target DNA on the plasmid.

1.1.2 Transformation and Selection

The transformation of the competent E. coli strain BL21(λDE3) by thepET-15b::mabA plasmid was carried out by thermal shock (Material andMethods). The effectiveness of transformation obtained is 5.9 103 CFU/μgof DNA. The weak effectiveness of transformation characterizing thestrains of B coli from which BL21(λDE3) is derived is noted.

The selection of the cells having incorporated the plasmid is carriedout thanks to the acquisition of resistance to ampicillin. In ourselection experiments, we preferred to use carbenicillin, a stableβ-lactain, rather than ampicillin which is known to be rapidly degradedby the β-lactainases secreted by the resistant bacteria.

1.1.3 Verification of the Sequence of the Cloned mabA Gene

In order to verify that no mutation was introduced during the PCRamplification stage of mabA, the sequence of the cloned gene wasanalyzed. No mutation was found; the cloned sequence is identical tothat carried by the original cosmid.

1.2. Heterologous Expression and Optimization

The optimization of the expression of a heterologous gene requires apreliminary small-scale study in order to determine the choice ofculture conditions and induction parameters (OD, temperature,concentration of the inducer and induction time). The development ofthese conditions allowed us to define the procedure to be followed inorder to obtain a sufficient overproduction of the protein which isvisible in SDS-PAGE. However, despite our efforts to reproduce theoverexpression on a larger scale, we did not succeed in producing theprotein MabA by the bacteria induced. The most plausible hypothesis wasthe loss of the plasmid, despite the maintenance of the cultures inmedium containing the antibiotic. Faced with this problem, the plasmidstability test in a dish (Material and Methods) offered us a rapid andreliable means of verifying, in the cultures before induction, thepresence of the target plasmid on the one hand, and the ability of thebacteria transformed, in culture, to express the heterologous DNA, onthe other hand.

The heterologous expression of mabA in E. coli proved particularlysensitive to the culture conditions which affect the stability of theplasmid. For optimal expression, it is important to fulfill twoconditions:

The transforming colonies must be fresh (coming directly from atransformation or a plating by stria from a liquid stock stored at −70°C).

The number of generations between the transformation of the bacteria bythe plasmid and the induction of the expression must be reduced to aminimum (avoiding intermediate cultures).

2. Purification of MabA

2.1 Solubility of the Overproduced Protein and Optimization

In order to establish the purification strategy, it is important to knowwhether the protein is produced in soluble form, or localized in theinclusion body (aggregates of proteins).

The small-scale tests to determine the solubility under optimuminduction conditions revealed certain interesting and unexpected points.The first is that the variations applied to the induction parameters(temperature, OD or duration of induction), aimed at improving thesolubility, do not seem to have significant consequences on thepreferential production of the protein MabA in such or such a form. Onthe other hand, we were surprised to note that the technique adopted forlysing the bacteria modulated the distribution of the protein betweenthe soluble and insoluble fractions. For example, cold sonication in areduced volume (concentration factor of the culture CF>20) is probablyresponsible for the precipitation of MabA in the pellet (insolublefraction). The effect of the high local temperatures engendered by theultrasonics could explain this phenomenon of aggregation-precipitationof MabA. The lysis of a bacterial suspension at a lower cell densitymakes it possible to avoid the precipitation of MabA during sonication.

A study carried out on the effects of ultrasonics on enzymes (Coakley etal., 1973) reveals that the cellular extracts prepared by ultrasonicdisintegration are sensitive to the damage caused by the free radicals,which are-probably generated by the ultrasonics, as well as by theeffect of high local temperatures. These authors show that the“damaging” effect during the lysis of the bacteria can be minimized bysonication at a high concentration in cells and in the presence ofcomponents of the medium such as the sugars (acting as “scavengers” ofradicals). These conclusions do not seem to be in agreement with ourresults which show that at a lower cell density during the lysis ofMabA, the protein is found in the “soluble” fraction. However no theorycan be advanced as to the activity of the protein under theseconditions.

After comparison of several lysis techniques on the desired productionscale, we opted for a lysis by lysozyme, followed by a freeze-thawcycle. According to this protocol, and under the induction conditionsadopted [OD600=0.8, 2 hours at 37° C., 0.8 mM IPTG], a large part of theprotein MabA is found in the soluble fraction.

2.2 Purification System

The obtaining of the protein MabA with a poly-His (H-MabA) tagfacilitates its purification. In fact, the high affinity of thehistidine residues for the metal ions makes it possible to use theimobilized metal ion affinity chromatography (IMAC) method. One of thematrices most used for its effectiveness is nickel-nitrilotriacetateNi-NTA-agarose (Qiagen). The NTA group has 4 chelation sites interactingwith 4 of the 6 coordination sites of the metal ion Ni. The imidazolenuclei of the histidine residues bind to the nickel ions on the Ni-NTAmatrix. The addition of imidazole molecules makes it possible, bycompetition with the histidine residues, to break the bonds between theproteins and the matrix, and to elute the bound poly-His protein. Theaffinity of a protein for the Ni-NTA-agarose matrix is a function of thenumber of histidine residues which it possesses and which are exposed tothe matrix. Thus, by adjusting the imidazole concentration, differentspecies of proteins having different degrees of affinity can be eluted.The very high affinity of the proteins having a poly-His tag for nickelmakes it possible to separate it from the majority of the proteinsco-produced by E. coli. Thus, if the binding of the protein H-MabAproves sufficiently specific, purification will be limited to the singlestage of affinity chromatography.

2.3 Purification of MabA in Native Conditions

The development of the conditions for elution of the protein MabA onNi-NTA-agarose resin is carried out on a small scale (50 μl), using theso-called resin sedimentation method in batches. Thanks to thistechnique, we were able to determine the different imidazoleconcentrations necessary for the elution of the protein MabA andelimination of the other proteins.

The purification adapted on a larger scale is carried out in open columnwith 500 μl of resin in suspension (Material and Methods). Moving frombatch purification to column purification required an additional stageof development.

The protein fractions corresponding to the different purification stagesare analyzed by SDS-PAGE. In the clarified lysate, the majority bandobtained between 30 and 43 kDa and corresponding to MabA, providesevidence of a fairly large overproduction of the protein in solubleform, more than 50% of the soluble proteins of E. coli. After the stageof binding of the proteins on the resin, the fraction containing thenon-bound proteins on the column is devoid of MabA, indicating aneffective binding of the protein H-MabA to the Ni-NTA matrix. Theelimination of other proteins weakly bound to the matrix (by thepresence of histidines dispersed in their sequence), is obtained afterextensive washings with 50 mM imidazole. An imidazole concentrationequal to 175 mM is required in order to elute the protein H-MabA aloneand in a very large quantity. The apparent masse of H-MabA deduced fromits electrophoretic migration is estimated at 35 kDa.

2.4 Problems of Precipitation of MabA During the Purification

During the purification, we noted that the protein MabA, eluted at avery high concentration, immediately precipitated in the tube. Thisbehaviour often observed for the proteins with a poly-His tag, isprobably due to non-specific protein-protein interactions due to thevery strong local protein concentration during the purification (TALONTMMetal affinity Resin—User Manual CLONTECH).

Attempts at solubilization of the protein eluted with detergents (TritonX-100, NP-40) proved to be in vain. It was therefore necessary tointervene before the elution of the protein. In order to prevent theprecipitation of the protein, we carried out a treatment before andafter purification. In order to verify whether the protein precipitates,the fraction which contains MabA after elution is centrifuged (5minutes, at 12,000 g) then the supernatant and the pellet are analyzedby SDS-PAGE (Material and Methods). The pre-purification treatmentconsists of adding mild “solubilizing” agents to the lysate. Afterpurification, the eluted protein is recovered directly in glycerol (50%final) (glycerol is a protective agent, much used for preserving theactivity of the enzymes).

Three conditions were tested:

10% (v/v) glycerol alone

10% (v/v) glycerol+0.1% (v/v) Triton X-100 (non-ionic detergent)

10% (v/v) glycerol+0.05% (v/v) (7 mM) β-mercaptoethanol (reducingagent).

The three processes made it possible to improve the solubility of theprotein MabA. It was noted however, during the use of 7 mMβ-mercaptoethanol, that H-MabA begins to be eluted at a much lowerimidazole concentration (50 M instead of 175 mM).

The solubilization of MabA in the presence of the three agents testedbeing comparable, we opted for the addition of 10% glycerol alone to thelysate.

2.5 Protocol Optimized for the Overproduction and Purification of H-MabA

The optimization of the conditions for overexpression and purificationof H-MabA allowed us to adopt the following protocol:

200 ml of E. coli/h-mabA on LB+CBC 50 μg/ml are cultured up toOD600=0.8;

Expression is induced with 0.8 mM IPTG for 2 hours, at 37° C.;

The bacteria are sedimented by centrifugation for 15 minutes at 10,000g, at 4° C. The pellet is taken up in 9 ml of lysis buffer (5 mMimidazole and 500 mM NaCl);

Lysozyme (0.5 mg/ml) and the protease inhibitors (0.113 mg/ml) areadded;

Freezing is carried out overnight at −70° C;

Thawing is carried out for 1 hour at ambient temperature and treated bythe DnaseI (5 μg/ml) and the RnaseA (10 μg/ml) in the presence of MgCl2(10 mM), 15 minutes at 4° C.;

The lysate is centrifuged at 3,000 g then at 10,000 g, and the solublefraction recovered;

The supernatant is centrifuged for 45 min at 44,000 g, at 4° C. and the“clarified lysate” recovered;

10% of pure glycerol (v/v) is added to the soluble fraction anddeposited on a mini-column (500 μl of Ni-NTA-agarose phase). Incubationis carried out for 1 hour at 4° C. under gentle stirring;

The phase is washed with 5×4 ml of elution buffer with 5 mM imidazole;

Pre-elution is carried out with 8×500 μl of 50 mM imidazole;

Elution is carried out with 8×500 μl of 175 mM imidazole;

Washing is carried out with 10×500 μl of250 mM imidazole;

The fractions containing the protein are collected according to theirconcentration and their purety. The protein is collected directly in anequal volume of pure glycerol, followed by dialysis against 50 mMpotassium phosphate buffer, pH 7.2, containing 50% glycerol and storedat −20° C.

Elution buffer: 50 mM potassium phosphate buffer, pH 7.8

2.6 Purification Yield

Thanks to the expression and purification system used, it was possibleto purify the protein H-MabA to homogeneity in a single stage. In orderto know approximately the concentration of the protein solution, itsultraviolet absorbance at 280 nm was determined. Knowing the absorbanceof the tyrosine and tryptophan residues of the protein (Material andMethods), the theoretical molar extinction coefficient of MabA wasdeduced (ε_(280 nm)=9530 M⁻¹cm⁻¹) and the molar concentration of thepurified solution was estimated at 40 μM.

The best purification yield (percentage of purified protein relative toall of the total proteins deposited on the column) obtained is 57%.Starting with 200 ml of culture, we obtained approximately 20 mg of pureprotein MabA (yield 100 mg/l of culture), which is very satisfactory.

3. Characterization of the Purified Protein MabA

3.1. Verification of the Peptide Sequence

The mabA gene cloned in pET-15b was sequenced, no mutation was found.The primary sequence of the wild-type protein MabA has 247 amino acids.The poly-histidine tag of the recombinant protein adds 19 amino acids toit (266 amino acids in total). The sequencing of the first 20 aminoacids of the overexpressed protein MabA was carried out (Biomerieux,Lyon). We were able to verify the identity of the protein on theamino-terminal part and detect the loss of the first methionine of thepoly-His tag. The elimination of the amino-terminal methionine fromproteins by post-translational proteolysis is very frequent in E. coli.

3.2. Control of the Purity of the Sample

Analysis by denaturing electrophoresis (SDS-PAGE) and Coomassie bluestaining of the eluate MabA shows a single band, indicating thehomogeneity of the preparation. The purity of the protein was alsoverified by SDS-PAGE after staining with silver nitrate. No contaminantprotein band is detected by this very sensitive development technique.

In order to determine the mass of the purified protein with precision,analysis by electrospray ionization mass spectrometry (ESI-MS) wascarried out.

3;3 Determination of the Molecular Mass

Mass spectrometry makes it possible to verify very rapidly that theprotein expressed has the expected mass. We analyzed a sample purifiedby electrospray ionization/mass spectrometry (ESI/MS), in collaborationwith B. Monsarrat (IPBS, Toulouse). On the type of instrument used, themolecular mass of a protein is determined with a precision of 0.01% (1/10,000). The mass of the protein MabA predicted from the gene sequence(taking account of the poly-His tag) is 27,860 Da. Analysis by ESI/MS indirect introduction reveals a majority mass of 27,728±2 Da.

The difference between the theoretical mass and the measured mass (131mass units) corresponds to the loss of the first methionine at theamino-terminal end, detected by the N-terminal sequencing of theprotein. The molecular mass of the purified protein H-MabA thusdetermined is 27,728 Da.

The migration of H-MabA in denaturing electrophoresis towards 35,000 Dacould be linked to the physico-chemical characteristics of the proteinand/or to its native form.

3.4 Determination of the Quaternary Structure of MabA by Gel Filtration

Exclusion chromatography makes it possible to determiner the native form(quatemary structure) of the protein in solution at a givenconcentration and under the defined conditions of pH and ionic strength.Thanks to this technique, it is possible to establish a relation betweenthe elution volume of the protein and its molecular weight, via acalibration curve. The calibration curve is deduced from the elutionprofiles of the standard proteins (Pharmacia).

The elution of the protein MabA (0.66 mg) was carried out under the sameconditions as those of the standard proteins. On the chromatogram, aneluted asymmetrical peak is observed towards the high molecular weights.The elution volume corresponding to the top of the peak indicates thatthe majority molecular mass (94.6 kDa) is comprised between 110,916 Daand 83,187 Da, corresponding to the tetrameric or trimeric form ofH-MabA, respectively. The slight shoulder distinguished on the profile(around 57.7 kDa) shows the presence, in a smaller proportion, of adimeric form (55,458 Da) of the protein. These results suggest thatthere is probably a dimer-tetramer equilibrium of the protein MabA.Study of the three-dimensional structure by molecular modelling of MabAfavours this hypothesis (see hereafter). It is however important tostress that the determination of the oligomeric structure by gelfiltration is dependent on the tested conditions and in particular theconcentration of the protein solution. The possibility of the presenceof the protein MabA in vivo in the tetrameric form will be discussedhereafter.

3.5 A Few Physico-Chemical Properties of MabA

Certain physico-chemical properties of MabA can be deduced from itspeptide sequence using one of the calculation programmes available onthe Internet (aBi). The sequence of 266 amino acids of the proteinH-MabA produced gives a calculated mass equal to 27729.37 Da. Itcorresponds to that determined by ESI/MS, to approximately 1 mass unit.The other characteristics are summarized in Table I hereafter. TABLE IPhysico-chemical characteristics of H-MabA Parameter Value Note Sequence266 amino acids Absence of the N- terminal methionine Molecular mass27,729 Da Verified by ESI/MS Molar extinction* 9530 M⁻¹ cm⁻¹ Low,presence of a coefficient ε _(280 nm) tryptophan and three tyrosines inthe sequence Absorbance* at 280 nm A_(280 nm) ^(0.1%) = 0.348 of a 0.1%solution (1 mg/ml) Isoelectric point Ip* 9.79 net charge (+) at neutralpH*estimated by calculation, from the peptide sequence.

4. Catalytic Activity of MabA

The attribution of a potential activity to a protein of unknown functionis often based on the similarity of sequence which it has with knownproteins. Examination of the primary structure of the protein MabAdemonstrates a strong identity with the sequence of the β-ketoacyl-ACPreductase FabG of E. coli (44% identity over 241AA), as well as with theβ-ketoacyl-ACP reductases of other bacteria or plants. This enzymaticactivity corresponds to one of the stages of the classic biosynthesisroute of fatty acids. The elongation of fatty acids by the mycobacterialsystem FAS-II involves the protein InhA, which catalyses theNADH-dependent enoyl-ACP reduction stage. The elongation system FAS-IIbeing comprised of several aggregated enzymes, it was logical toenvisage the presence of the protein MabA combined with InhA in the sameenzymatic complex. A strong argument in favour of the involvement ofMabA and InhA in the same metabolic route rests on the operonorganization of the mabA and inhA genes in M. tuberculosis. The genesinvolved in the biosynthesis of fatty acids are often grouped into“clusters” as for example in E. coli (Rawlings & Cronan, 1992) and inVibrio harveyi (Shen & Byers, 1996).

Detecting the β-ketoacyl reductase activity of the purified protein MabAis the first stage of its characterization as potential partner of InhAin the biosynthesis of fatty acids.

4.1 Enzymatic Characterization of the Protein MabA

4.1.1. Demonstration of the Catalytic Activity of MabA

4.11.1. Description of the Enzymatic Test

The activity of the purified-protein H-MabA was first tested in thepresence of the only commercial β-ketoacyl-CoA, acetoacetyl-CoA, andNADPH as electron donor. The addition of pure MabA to the substratestriggers the reaction. The evolution of the reaction is monitored for 5minutes by measuring the reduction in absorbance at 340 nm, expressingthe disappearance of the NADPH co-substrate in favour of its oxidizedform NADP⁺ (which does not absorb at this wavelength).

Under the standard enzymatic test conditions defined (see Material andMethods), H-MabA is capable of reducing acetoacetyl-CoA. The purifiedprotein H-MabA is therefore functional: it corresponds to a β-ketoacylreductase (KAR: keto-acyl reductase). The presence of the poly-His tagin N-terminal position does not seem to impede its activity.

The substitution of NADPH by NADH at the same concentration in thekinetics test leads to a total loss of the activity. The protein MabA istherefore strictly NADPH-dependent. The presence in the peptide sequenceof MabA of an NADP(H) binding unit confirms this result. The KARs ofother organisms are most often NADPH-dependent and have a strictspecificity for the nucleotide coenzyme.

4.1.1.2 Parameters Affecting the Activity of the Protein MabA

The activity of an enzyme is directly affected by the concentration ofits substrates, but also by parameters such as the nature of the buffer,pH, the ionic strength, temperature. In order to optimize the reactionconditions, we studied the effect of the pH and ionic strength on theactivity of MabA.

Effect of the pH

We evaluated the effect of the pH on the enzymatic activity of MabAusing sodium phosphate buffer solutions with a pH of 5.0 to 8.0 in thereaction medium. Comparison of the results for the chosen pH range showsthat the optimum activity of MabA is obtained for a pH equal to 5.5.However, at an acid pH (5.0 to 6.5), the NADPH is very unstable and isoxidized spontaneously, which leads to a variation in absorbance overtime in the absence of enzyme. We therefore decided to work at pHs closeto physiological pH (between 7.0 and 7.5), for which the base line has anegligible gradient compared with the catalysis gradient (less than5-10%). Other β-ketoacyl-ACP reductases have an acid optimum pH (around6.0-6.5) [(Shimnakata & Stumpf, 1982); (Caughey & Kekwick, 1982)].

If MabA has a better activity at pH 5.5, this is probably linked to aprotonation event involved in the binding of the substrates or in thecatalysis. This event could concern two His residues of the protein, H46and H247 (the pKa of the imidazole nucleus of the histidine residue isequal to 6.0-6.5), potentially involved in the active site, according tothe structural model of MabA.

Effect of the Ionic Strength

The MabA activity tested is constant for phosphate buffer concentrationsvarying between 20 and 100 nM. We opted for an 80 mM buffer, pH 7.0.

Effect of Dilution

The enzymatic tests requiring a preincubation of H-MabA over timerevealed that the catalytic activity decreases rapidly if the enzyme isincubated at a low concentration (molar concentration <1 μM). Theinactivation by dilution of the β-ketoacyl-ACP reductases of E. coli andof plants (Brassica napus, Persea americana) has already been reported(Schulz & Wakil, 1971); (Sheldon et al., 1990); (Sheldon et al., 1992)].

4.1.2. Determination of the Kinetic Parameters of MabA

The characterization of an enzyme generally comprises the determinationof the maximum reaction velocity, Vmax and of the “Michaelis constant”,Km, for each substrate. Knowledge of these parameters proves very usefulfor biochemical studies (comparison of the affinity for differentsubstrates, interaction with other molecules, comparison of isoenzymesof different organisms) and in particular for defining the effectivenessof inhibitors or activators of the enzyme.

4.1.2.1 Measurement of the Km for NADPH

Determination of the kinetic parameters Vmax and Km begins with theestimation of the Km value, by testing two concentrations of substrate,one low and the other high. The initial reaction velocities are thendetermined for a preferably wide range of concentrations in substrate,if possible covering from Km/2 to 5 Km. We plotted the straight lineS/v=f (S) or 1/v=f(1/S) in order to visualize the data, then we comparedthe Km and Vmax values calculated by this method and those obtained bythe least error squares method. The values obtained are the average ofthree manipulations. The determination S/v=f (S) produces results closeto those obtained by the least error squares method. The value of Kmobtained for NADPH, 39 μM, is approximately five times greater than thatof the protein InhA for its cofactor NADH (8 μM). This higher Kmprobably reflects a lesser affinity of MabA for its coenzyme. The Km'sof the β-ketoacyl reductases of other organisms for their cofactor areof the same order of magnitude as that obtained for MabA.

4.1.2.2 Measurement of the Km for Acetoacetyl-CoA

The Km for the acetoacetyl-CoA, determined in the presence of NADPH, is1582 μM. This relatively high Km is much greater than the Km describedfor other β-ketoacyl-ACP reductases of plants. The fact that MabA has ahigher Km than these enzymes which belong to of synthesis systems denovo, therefore specific to short chain substrates would suggest thatMabA could have a preference for substrates longer than 4 carbons. Studyof the specificity of MabA for substrates with a longer hydrocarbonchain thus seemed to us doubly important, on the one hand in order tobetter characterize the enzymatic activity of this protein and on theother hand in order to compare the substrate-specificity of MabA andthat of InhA. The protein InhA was shown to be specific to long chainsubstrates (12-24 carbon atoms), exhibiting no activity in the presenceof the substrate with 4 carbons (crotonoyl-CoA), even at 8 mM (Quemardet al., 1995).

4.1.3. Determination of the Kinetic Constants for the Long ChainSubstrates

The use of long chain substrates (C8 to C20) imposes constraints linkedto their critical micellar concentration (CMC). The long chain acyl-CoAsare amphiphilic compounds and only form true solutions at allowconcentration. Beyond the CMC, some of the molecules form micelles andthe monomer concentration is fixed at the CMC, therefore different fromthe total concentration. It was therefore important to use solutionswith concentrations below the CMC. In a study of the physical propertiesof acyl-CoAs (Constantinides & Steim, 1985), the CMC's of aqueoussolutions of palitoyl-CoA (C16-CoA) and stearoyl-CoA (C18-CoA)determined are respectively 70 and 12 μM. The presence of anunsaturation (in position 9) in the case of oleyl-CoA (C18:1-CoA) raisesits CMC to 33 M. The presence of a ketone function on the chain would intheory have a similar effect relative to the CMC. Using these data, weattempted to prepare solutions of β-ketothioester the concentration ofwhich was above the CMC. The stock solutions used for the kinetics testsare 400 μM and 100 μM for the C8 and C12 β-ketothioesters, respectively.

4.1.3.1. Measurement of the Km for the C8 and C12 β-Ketoacyl-CoAs

We measured the kinetic parameters of MabA for β-ketooctanoyl-CoA (C8)and β-ketododecanoyl-CoA (C12). The protein has a Km (60 μM) for the C8substrate 25 times lower than that of the C4 (1582 μM). The C12derivative also proves a much better substrate (Km of 9 μM). There also,the values obtained by the Hanes method and that of the “least errorsquares” method are similar. We calculated the Km/Vrmax ratio whichreflects the affinity of the enzyme for its substrates. Kn/Vmax becomeslower as the substrate chain length increases. This correlation is duenot only to the lower Km values, but also to higher Vmax values for thelonger chains.

The kinetic constants Km and Vmax for C16 and C20 were determined. Forthose β-ketoacyl-CoAs with more than 12 carbon atoms, problems ofinhibition by the substrate were encountered, also described in the caseof the use of substrates of InhA of a size greater than C16. Wetherefore compared the initial reaction velocities at the sameconcentration (2 μM), in the presence of different β-ketoacyl-CoAs (C4to C20). In order to measure the activity, it was necessary to usesolutions of enzymes at different concentrations for the variousβ-ketothioester substrates. The protein MabA has a considerablepreference for the 12-carbon substrate compared with the shortsubstrates, and the C16 and C20 β-ketothioesters prove to be substratesat least as good as the C8. The reduction in the reaction velocityobserved for the long chain of β-ketoesters could be linked to their lowsolubility (in the case where the real concentration of free moleculeswould be less than 2 μM).

4.1.3.2. Substrate Specificity and Involvement in an Elongation Route?

Although it has an activity in the presence of 4-carbon β-ketoacyl, theprotein MabA nevertheless shows a clear preference for the C12-C16substrates. The affinity of MabA for the long chain hydrocarbonsubstrates is compatible with the size and hydrophobic nature of thesubstrate-binding pocket. The protein InhA itself has a slightlydifferent affinity, with a preference for longer C16-C24 substrates(Quémard et al., 1995). The enzymatic properties of MabA and InhA, inparticular their specificity for medium to long chain substrates, goesin the direction of their belonging to the same fatty acids elongationsystem, FAS-II, which is itself specific to C12-C18 substrates.

The specificity of InhA substrate differs from that of the enoylreductases of the type II systems of Spinacea oleracea (Shimakata &Stumpf, 1982)) or of E. coli (Weeks & Wakil, 1968), which have apreference for C6 and C8 substrates. Moreover, the β-hydroxyacyldehydratase of the type II system of E. coli (Birge & Vagelos, 1972) isspecific to short-chain substrates (C4 to C12), whereas it is only veryslightly active in the presence of C16 substrate. These data emphasizethe specificity of particular substrates of the mycobacterial FAS-IIsystem.

4.1.4. MabA and ACP-Dependence?

The enzymatic complex containing hihA which we identified as theelongation system FAS-II, apart from its specificity for the C12-C18substrates, has the property of being ACP-dependent. The ACP-dependenceof the protein InhA is illustrated by its much more marked affinity forthe substrates derived from ACP (the Km for octenoyl-ACP is 2 orders ofmagnitude smaller than that for the derivative of C8 CoA). Determiningthe preference of MabA for ACP derivatives requires the synthesis ofthese (non-commercial) derivatives and comparison of the kineticconstants with those- of the CoA derivatives. The KARs of plants areACP-dependent, a property which was correlated to their belonging to atype II system. The numerous arguments in favour of MabA belonging toFAS-II strongly suggest the ACP-dependence of β-ketoacyl reductase.

Conclusion

After development of the overproduction of the protein MabA inEscherichia coli and purification, we carried out an enzymatic study ofthis protein. Thus, we showed that its catalytic activity corresponds tothe NADPH-dependent reduction of β-ketoesters, which corresponds to oneof the stages of the fatty acid elongation route. Determination of theactivity of MabA in the presence of substrates with several chainlengths made it possible to show the preference of this enzyme forsubstrates of a size greater than or equal to 12 carbon atoms, inaccordance with its potential involvement in a fatty acid elongationsystem. We therefore sought the protein MabA in the FAS-II enzymaticcomplex containing InhA, and studied the involvement of MabA in theelongation activity of this system.

5. Contribution of Molecular Modelling to the Study of the Protein MabA

Molecular modelling makes it possible to access a set of informationconcerning the structural characteristics of the protein, thearchitecture of the catalytic site, but also to assess the possibilitiesof interaction with ligands (substrates, inhibitors, affine molecules).The production of the three-dimensional model of the protein MabA ispresented below.

5.1. Search for Proteins Having a High Sequence Similarity With MabA

A search for peptide sequences similar to that of MabA (M. tuberculosis)in data banks with Psi-blast software (Altschul et al., 1997) showedthat β-ketoacyl-ACP reductases existed having a high level of identitywith MabA (87%, 84%, 69%, respectively) in other mycobacterial species(avium, smegmatis, leprae). Proteins homologous to MabA, called FabG,are also present in other organisms, essentially bacteria (for examplein Streptomyces ceolicolor, 57% identity) and plants. However, noβ-ketoacyl-ACP reductase structure has ever been resolved. Producing amolecular model of MabA was therefore of interest in the study of FabG.

5.2. Production of the MabA Model

The structural modelling of MabA was carried out using the programmeModeller 4 (Sali & Blundell, 1993). The model is based on the structuresof proteins crystallized in complex with NAD(P)(H) and having thehighest level of identity and lowest probability score (E) with MabA.These “support” proteins, selected using Psi-blast software (Altschul etal., 1997) in the main protein structure data bank, the PDB (ProteinData Bank, (Berman et al., 2000)), are: PDB2HSD (34%/NAD); PDB1YBV(33%/NADPH); PDB2AE2 (29%/NADP); PDB1FMC (28%/NADH); PDB1CYD (28%/NADPH)and PDB1BDB (27%/NAD). These proteins, of very diverse origin, allcatalyze either the reduction of a carbonyl (such as MabA), or thereverse reaction. The alignment of sequences used for the modelling wascarried out by considering the well-preserved regions between MabA andthe supports, on .the one hand, and between MabA and the other knownβ-ketoacyl-ACP reductases (FabG), on the other hand. In order to verifythat the model is energetically stable, two programmes were used, TITO(Labesse & Momon, 1998) and Verify-3D (Luthy et al., 1992), whichproduced satisfactory scores.

The monomeric structure produced by the MabA model indicates that theprotein belongs to the α/β structural superfamily, with six α helicesand seven β strands. It should be noted that the β6-α6″ loop comprisestwo helices called α6 and α6′. MabA possesses a single domain, thetopology of which is similar to Rossmann folding (β/α)₆ (Rossmann etal., 1974), typical of the dinucleotide-diphosphate-binding proteins(DDBP) (Persson et al., 1991). However, in contrast to the DDBP with twodomains, there is no symmetry, since the helices of the C-terminalmoiety (α4, α5) are longer than the secondary structures of theN-terminal part. These characteristics, as well as the presence of anadditional strand (β7), are typical of the RED(Reductase/Epimerase/Dehydrogenase) proteins superfamily (Labesse etal., 1994). The presence of a single cysteine (C60), probably buried, inMabA excludes the possibility of formation of an intra- or inter-chaindisulphide bond within the protein.

The bound NADPH cofactor is found in an extended conformation resting onthe C-terminals ends of the β1-β5 strands which form a leaf. The β2strand of the RED proteins which is involved in the binding of theribose linked to the adenine of the cofactor, has, in the MabA sequence,the unit [* * * xxr]

, specific to NADP(H)-dependent enzymes (Labesse et al., 1994). This isin agreement with the enzymatic data showing the strict specificity ofMabA for NADPH and indicates that the additional phosphate is probablyimportant for the stabilization of the cofactor in satisfactoryorientation for the catalysis. The positively charged residue R47,forming part of the unit [VAVTHR] of the strand β2, is probably involvedin the interaction of the protein with the phosphate, by electrostaticbonds.

*: hydrophobic residue, x: any amino acid. In capital and small lettersthe strictly preserved residues and those most frequently encountered,respectively.

As in the other RED proteins, the binding site of the substrate of MabAis probably delimited by the C-terminal ends of the strands β4, β5, β6,β7 and the helices α4, α5, α6 (α6, α6′, α6″) (FIG. 5.18; (Labesse etal., 1994)); the nicotinamide part of NADPH, involved in the ionexchanges, is oriented towards the bottom of the cavity. The residues ofthe active site which are very well preserved, and constitute in partthe signature of RED proteins, are present in the catalytic site ofMabA: the catalytic triad, S140,Y153, K157 and N112, T188.

5.3.Relation Between Structure and Function of MabA

According to the atomic coordinates of the MabA model, the singletryptophan (W145), situated at the level of the β5-α5 loop, is probablyinvolved in the catalytic pocket. The latter appears very hydrophobicbecause of the involvement of the C-terminal arm (rich in hydrophobicresidues) in the structure of this pocket on the one hand, and by thepresence, in addition to W145, of residues such as I147 and F205, on theother hand. In the proteins FabG of other organisms and specific toshort chain substrates, the latter two residues are replaced by morepolar residues, Asn (for I147) and Thr, Gln or Asn (for F205). Thespecificity of MabA for long chain substrates is very probably linkedwith the hydrophobic character of the catalytic pocket which thusconstitutes a favourable environment for receiving aliphatic longchains, a structure-function relation between the hydrophobicity and thesize of the substrate-binding pocket and the affinity for long chainmolecules has already been demonstrated for the protein InhA (Rozwarskiet al., 1999), which also forms part of the REDs.

The superposition of the MabA model on the crystalline structure of InhA(ternary complex C16 InhA-NAD⁺-substrate, (Rozwarski et al., 1999);PDB1BVR) reveals that the substrate-binding pockets of the two proteinshave similar sizes, in accordance with their affinity for substratespossessing similar chain lengths (C₁₂-C₂₄ for InhA, C₈-C₂₀ for MabA;[(Rozwarski et al., 1999); (Quémard et al., 1995)]. However, the bindingpocket of the enoyl reductase InhA is still more hydrophobic than thatof MabA, which could explain the slight shift in the specificity ofsubstrates between InhA (maximum specific activity in the presence ofC₁₆, (Quémard et al., 1995)) and MabA (maximum specific activity in thepresence of C₁₂).

The alignment of MabA sequences with the support proteins and with allof the known proteins FabG indicates that the amino-terminal end is notpreserved; this region “floats” to the outside of the protein and doesnot correspond to a defined secondary structure. This suggests that thisdomain of the protein can tolerate variations, and that it is notimportant for the function of the protein. Experimental data inagreement with this proposition are provided by study of the catalyticactivity of H-MabA. The protein comprises an NH₂-terminal poly-histidinetag the presence of which does not seem to affect the catalyticactivity.

5.4. Quaternary Structure of MabA

Due to their secondary structures and tertiary characteristics, all theRED proteins described are dimeric or tetrameric (dimer of dimers). TheC-terminal region of MabA, corresponding to the α6-β7 loop and the β7strand, has a very high similarity with the equivalent region of theknown tetrameric REDs, in particular with that of PDB2HSD for which itwas shown that this region was involved in the dimer-dimer interface ofthe heterotetramer (Persson et al., 1991). The second interface betweentwo monomers in PDB2HSD involves the helices α4 and α5. The preservationin MabA of the C-terminal end and the presence of hydrophobic aminoacids at the surface of the helices α4 and α5 suggest that MabA istetrameric. These results are in agreement with the exclusionchromatography analysis, suggesting an equilibrium between the dimericand tetrameric forms of MabA. Analysis of the monomer-monomer anddimer-dimer interfaces in a tetrameric model of MabA could make itpossible to reinforce this conclusion.

5.5. Interaction With Antibiotics

It has been shown that the active form of the INH which inhibits theprotein InhA would be an isonicotinoyl-NAD radical or anion (Rozwarskiet al., 1998). These authors have suggested that the isonicotinoyl-NADadduct is formed in the catalytic site of InhA, whilst Wilming andJohnsson have shown that its formation can occur in the absence of theenzyme (Wilming & Johnsson, 1999). Thus, doubt remains as to the exacteffect of the INH on IhA in vivo. The superposition of the MabA model onthe structure of the binary complex InhA-isonicotinoyl-NAD (PDB1ZID)shows that there is no incompatibility with the binding, in the activesite of MabA, of molecules such as isoniazid or ethionamide. Similarlyfor the protein InhA, the isonicotinoyl-NADP adduct could a priori befixed on MabA, once it is formed. However, in the case of the adductbeing formed within the catalytic site, it cannot be foreseen whetherthe isoniazid would have an appropriate orientation and could interactwith the cofactor NADPH. In all cases, an inhibition of the activity ofMabA by INH must be verified biochemically, as the model does not allowa precise teaching on the topology of the lateral chains of the activesite.

6. Inhibition of the Activity of MabA

We tested the effect of isoniazid on the β-ketoacyl reductase activityof MabA by adopting experimental conditions similar to those making itpossible to observe an inhibition of the activity of InhA. The proteinMabA (150 nM) is preincubated for 2 hours in the presence of 100 μM or 2mM isoniazid, 100 μM NADPH and 1 μM MnCl₂. In the presence of 100 μM ofINH, the activity of MabA, demonstrated in the presence ofacetoacetyl-CoA, is inhibited by 44±3% compared with the control withoutINH and the addition of 2 mM of isoniazid, leads to an inhibition of62±6%. The fact that total inhibition of the activity of MabA is notobserved even in the presence of 2 mM isoniazid could be explained by avery slow oxidation of the isoniazid by the Mn³⁺ ions under theconditions used (in the absence of a catalyst such as KatG), andtherefore a concentration in active form of the antibiotic which is notproportional to the starting concentration of isoniazid. Thisexplanation assumes that MabA is inhibited by a mechanism similar tothat described for InhA. It should be recalled that the inhibitionmechanism of the protein InhA by isoniazid requires at a minimum thepresence of the cofactor NADH, Mn²⁺ ions and molecular oxygen. The Mn²⁺ions would be oxidized to Mn³⁺, which, in turn catalyze the oxidation ofthe isoniazid. Thus, we tested the effect of the absence of MnCl₂ orNADPH on the inhibition of MabA by INH. In the absence of MnCl₂ in thereaction, a non-significant reduction in the activity of the enzyme isobserved, indicating that the Mn²⁺ ions are necessary in order to obtainan effect of the INH. Determination of the involvement of NADPH in theinhibition process of is more difficult to achieve, as preincubation ofthe protein MabA in the absence of this cofactor leads to a considerablereduction (−74%) in the activity after preincubation for 2 hours withoutantibiotics. It was therefore not possible to evaluate the involvementof NADPH in the inhibition by isoniazid.

Our results show that the activity of the protein MabA is inhibited byisoniazid, and suggest that the action mechanism of this antibiotic onMabA would cause the intervention of Mn²⁺ ions. Given the structure andfunction homology of the two proteins, it is probable that theinhibition mechanism is analogous to that of InhA, passing through theformation of an isonicotinoyl-NADP⁺ adduct.

In mycobacteria, a mutation or overexpression of the inhA gene leads tocross-resistance to the two antituberculous agents isoniazid andethionamide (ETH). Ethionamide is probably also a prodrug since theinhibition of the protein InhA by this antibiotic in its native form hasnot been observed in vitro. However, the mode of activation of ETH isnot known. We nevertheless tested the effect of ethionamide on theactivity of InhA, under the experimental conditions of inhibition byINH, in the presence of MnCl₂ and NADH. In the presence of 100 μM ofETH, the activity of InhA is unchanged. The same result is obtained onMabA. It was not possible to test higher concentrations of antibioticsdue to the strong absorbance that it has in the wavelength region usedfor the enzymatic tests. Nevertheless, the conditions for oxidation ofINH in vitro adopted for the ethionamide test do not seem to be thoserequired for the activation of ETH. The fact that thecatalase-peroxidase KatG, which accelerates the oxidation of isoniazidis not the activator of ETH [(Johnsson et al., 1995); (Basso et al.,1996)] is in agreement with this conclusion. On the other hand, ifoxidation of ETH is required for its action on its targets(s), theoxidation of a thioamide function proves more difficult than that of ahydrazide function (INH) and should require an oxidizing agent strongerthan Mn³⁺ ions.

7. Conclusion and Discussion

Study of the three-dimensional structure of MabA by molecular modellingmade it possible to show that the protein has a single domain, with asecondary structure of α/β type, and that it belongs to the REDstructural superfamily (reductases/epimerases/dehydrogenases). Theprotein MabA possesses the specific unit of the proteins binding NADP(H)and a substrate-binding pocket the size and hydrophobicity of whichpromote the reception of long chain β-ketoesters. These structural dataprovided by the MabA model are in agreement with the biochemical resultsobtained previously. The MabA model indicates that the tryptophan (Trp)residue, situated at the level of the β5-α5 loop, would be involved inthe substrate-binding pocket. Thanks to the uniqueness of this Trpresidue, it was possible to carry out fluorescence spectroscopyexperiments. They made it possible to validate the MabA model,confirming the involvement of Trp and at least one of the two Mets ofthe C-terminal end in the substrate-binding pocket on the one hand, andthe specificity of MabA for long chain substrates on the other hand. Thesuperposition of the MabA model with the crystalline structure of InhA(in complex with NAD⁺ and the C16 substrate, (Rozwarski et al., 1999)reveals that the two proteins have substrate binding sites of equivalentsize and more hydrophobic than their homologues of other organisms,involved in a synthesis de novo. This confirms the hypothesis ofco-involvement of MabA and InhA in the same fatty acids elongationcomplex.

The MabA model suggests that the protein in solution is tetrameric,which is in agreement with the result of the gel filtration experiments,having suggested that there was, under the experimental conditionstested, a dimer-tetramer equilibrium of MabA. However, the combinationof MabA with InhA, each in the tetrameric form in the FAS-II complex, isincompatible with the estimated size of the system. Thus, as InhA andMabA have similar topologies, it could be postulated that these twoproteins form a heterotetramer within the FAS-II complex. In order totest this hypothesis, the molecular modelling of an MabA-InhAheterotetramer complex, using tetrameric RED proteins as supports, canbe carried out. In order to confirm the possibility that InhA and MabAcan be combined in complex, chemical bridging between the two proteinscan be attempted in the presence of their respective cofactors.

Knowledge of the three-dimensional organization given by the modelsuggests a possible interaction between MabA and isoniazid. We were ableto show, by enzymatic studies, that the activity of MabA was effectivelyinhibited in vitro by this antibiotic and that the inhibition mechanismof MabA is probably comparable with that described for the protein InhA.

II) Material and Methods

M.1. Overexpression of the mabA Gene in E. Coli

M.1.1. Construction of the pET-15b::mabA Expression Plasmid

The mabA gene of M. tuberculosis was cloned between the NdeI and Xhosites of the pET-15b plasmid, downstream of a sequence coding for 6histidines.

M. 1.2. Transformation of BL21(λDE3) E. coli by pET-15b::mabA Plasmid

After preparation of competent bacteria of E. coli BL21(λDE3) (Sambrooket al., 1989), an aliquot (100 μl) is incubated in the presence of thepET-15b::mabA plasmid (39 ng) for 30 minutes in ice. The transformationis carried out by thermal shock (90 seconds at −42° C., then 2 minutesin ice). LB medium is then added and the suspension is incubated for 45minutes at 37° C. under stirring (250 rpm) before being plated onLB-agar dishes containing 50 μg/ml of carbenicillin. Incubation at 37°C. for approximately 18 hours makes it possible to obtain medium tolarge-sized colonies.

M.1.3. Induction of the Expression of the Target Gene

Four medium-sized colonies are used for seeding four cultures of 50 mlin LB medium+carbenicillin. The turbidity of the medium is measured byspectrophotometry at 600 nm hourly until the optical density reaches 0.8(middle of the exponential growth phase), i.e. after incubation forapproximately 4 hours. The expression of the mabA gene is then inducedwith 0.8 mM of IPTG for 2 hours at 37° C., then verified by SDS-PAGE. Analiquot of non-induced culture is preserved and will serve as negativecontrol of the induction.

M.1.4. Verification of the Overexpression

Once the expression of mabA is induced, an aliquot of 100 μl of cultureis analyzed in order to check the expression of the gene. Aftercentrifugation (5 minutes at 12000 g), the bacterial pellet is taken upin charge buffer (Laemnmli, 1970) in order to be applied to 12%polyacrylamide gel under denaturing conditions.

M.1.5. Small-Scale Protein MabA Solubility Test

The bacteria (10 ml) are collected by centrifugation for 5 minutes at3000 g, at 4° C. The pellet is resuspended in potassium phosphate buffer(100 mM, pH 7.2) in 1/20 of the initial volume of the culture. Thesuspension is sonicated using a microprobe (Vibracell, Bioblock), usingfour pulses of 10 seconds interspersed with recovery times of 40 seconds(duty cycle: 60%, microtip limit: 5). The total extract obtained iscentrifuged for 5 minutes at 12000 g, at 4° C. The presence of theprotein MabA in the fractions corresponding to the total (soluble)supernatant and (insoluble) pellet is analyzed by SDS-PAGE (12%polyacrylamide).

M.2. Purification of MabA

All the stages are carried out at a low temperature (0-4° C.), in orderto reduce the action of the proteases.

M.2.1. Preparation of the Bacterial Lysate

All of the cultures (4×50 ml) are collected by centrifugation (15minutes at 16000 g, at 4° C.) then washed (50 mM potassium phosphatebuffer, pH 7.8). The pellet obtained (0.9 g/200 ml of culture) is takenup in 9 ml of lysis buffer (50 mM potassium phosphate buffer, pH 7.8containing 500 mM of NaCl and 5 mM of imidazole). Before freezing thesuspension at −70° C. (overnight), a mixture of protease inhibitors(0.113 mg/ml, see below) and lysozyme (0.5 mg/ml) are added to it. Thesuspension is thawed, under gentle stirring, at ambient temperature,then treated with DNaseI (5 μg/ml) and RNaseA (10 μg/ml) in the presenceof 10 mM MgCl₂ for 15 minutes at 4° C., under gentle stirring. The wholebacteria and the debris are eliminated by centrifugation (15 minutes at3000 g, at 4° C.). A last ultracentrifugation at 44000 g, 45 minutes at4° C. , makes it possible to eliminate any insoluble material. 10% (v/v)of glycerol is added to the supernatant (clarified lysate) before beingloaded on the column.

Mixture of Protease Inhibitors: leupeptin (chymotrypsin inhibitor):0.0023 g/l soybean (reversible trypsin inhibitor):  0.02 g/l TLCK(irreversible trypsin inhibitor): 0.0518 g/l Aprotinin 0.0016 g/lPepstatin (pepsin-like inhibitor) 0.0011 g/l PMSF (irreversiblechymotrypsin inhibitor) 0.0362 g/lNote:In these experiments, the EDTA (metal-dependent protease inhibitor) isomitted from the mixture of protease inhibitors, because of its abilityto chelate nickel ions during purification on an Ni-NTA column.

M.2.2. Purification of H-MabA in a Minicolumn

In an empty minicolumn (total volume 7.5 ml in polypropylene, Polylabo),500 μl of Ni-NTA agarose resin (QIAGEN) (i.e. 1 ml of 50% suspension)are washed with 4 times 25 ml of lysis buffer*. Four ml of clarifiedbacterial lysate (approximately 15 mg of total protein) are incubatedwith the Ni-NTA-resin under gentle stirring, for 1 hour at 4° C. Thematerial not bound to the resin is recovered by decantation, then by“washings” with 32 CV (column volumes) of lysis buffer. The remainder ofthe contaminant proteins is eluted with 8 CV of buffer with 50 mMimidazole. The protein MabA is eluted by 8 CV of buffer with 175 mMimidazole. The resin is then cleaned with 10 CV of buffer with 250 mMimidazole and recovered directly in pure glycerol in order to have 50%(v/v) of final glycerol. These precautions are necessary in order toavoid the precipitation of MabA at the column outlet.

Note: all the buffers used here contain 50 mM of potassium phosphate pH7.8 and 500 mM of NaCl.

lysis buffer: 50 mM potassium phosphate buffer, pH 7.8 containing 500 mMNaCl and 5 mM of imidazole.

M.2.3. Dialysis of the Solution of Purified H-MabA

The solution of protein MabA after purification, recovered in 50% (v/v)glycerol, is dialyzed twice for 1 hour against 40 volumes of 50 mMpotassium phosphate buffer pH 7.2 containing 50% (v/v) of glycerol, at4° C., in a dialysis tube (cutoff threshold 8-10 kDa, Spectra/Por,Spectrum) previously boiled in a solution of 1 mM EDTA in order toeliminate traces of heavy metals, then rinsed with osmosis-purifiedwater. The dialysate is then aliquoted and stored at −20° C.

M.2.4. Determination of the Quantity of Purified Protein by U.V.Spectroscopy

We estimated the concentration of the solutions of protein purifiedaccording to the Beer-Lambert law (OD=ε1C*) with its theoretical molarextinction coefficient and its absorbance at 280 nm.

ε: molar extinction coefficient, 1: length of the optical path and C:molar concentration.

Theoretical Determination of the Molar Extinction Coefficient (DeducedFrom the Protein Sequence)

The molar extinction coefficients (MEC) of the proteins are calculatedaccording to Gill and Von Hippel (Gill & von Hippel, 1989) (in thepresence of 6M guanidine chloride at pH 6.5). The relation is then thefollowing:

MEC=(a * ETyr)+(b * ETrp)+(c * ECys), where a, b and c are respectivelythe number of residues, and Eaa their molar extinction coefficients. At280 nm, they are respectively equal to: ETyr=1280 ETrp=5690 ECys=120

The MEC (ε) of MabA at 280nm is 9530 M⁻¹cm⁻¹ and does not take accountof the single Cys residue.

M3. Study of the Properties of MabA

M.3.1. Determination of the Mass of H-MabA by ElectrosprayIonization/Mass Spectrometry (ESI/MS)

A pellet of 2 mg of purified protein H-MabA, precipitated in bufferwithout glycerol, is washed 5 times with water (centrifugation for 5minutes at 12000 g). 200 μl of acetonitrile/water mixture (50/50)+0.1%(v/v) TFA are added, then the whole mixture is vortexed and centrifugedfor 2 minutes at 12000 g. An equal volume of a methanol/water mixture(50/50)+0.5% (v/v) acetic acid is added to an aliquot of thesupernatant, the mixture is vortexed then kept for 2 hours at 4° C.before being centrifuged for 2 minutes at 12000 g. 60 μl of thesupernatant are introduced into the source of the spectrometer via asyringe pump (HARVARD), at a flow rate of 5 μl/min, in order to beanalyzed by electrospray ionization/mass spectrometry (ESI/MS) on aFinnigan MAT device (TSQ 700). The parameters of the ESI sourcecorrespond to a 5 kV power supply, a temperature of the intermediatecapillary of 250° C. and 40 psi for the nitrogen (nebulization gas).

M3.2. Determination of Native Size by Gel Filtration

FPLC experiments were carried out with the BioCAD SPRINT system(PerSeptive Biosystems, Cambridge, Mass.). A Sephacryl S-100 HR column(HiPrep™ 16/60 Sephacryl High Resolution, Pharmacia) was equilibratedwith 1 CV of 50 mM potassium phosphate buffer, pH 6.8 containing 100 mMNaCl. Five standard proteins of known molecular masses, diluted in thissame buffer, were applied to the column (0.5 to 1 mg of each protein):alcohol dehydrogenase (150 kDa), bovine serum albumin (BSA, 67 kDa),ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and RibonucleaseA(RNaseA, 13.7 kDa). Two successive elutions were carried out with adifferent combination of 3 standard proteins in order to obtain a betterresolution of the peaks, and the profiles at 280 nm were superimposed.The calibration curve was obtained by plotting the elution volume ofeach standard protein as a function of the logarithm of the molecularmass. A solution of H-MabA at 1.1 mg/ml (0.66 mg of loaded protein) isapplied to the column and eluted sunder the same conditions as thestandard proteins. The molecular mass of H-MabA is estimatedwith-reference to the calibration curve.

M.4. Enzymatic Study of MabA

M.4.1. Calibration of the Solutions of Reagents

Determination of the kinetic parameters for the different substraterequires enzymatic test conditions which can be reproduced from onemanipulation to another. The concentrations of the solutions ofβ-ketoester substrate of CoA and cofactor (NADPH) are thereforedetermined before use. The reagent to be calibrated (for exampleβ-ketoester of CoA) is added at a concentration considerably lower thanthat of the second substrate (NADPH). The reaction is triggered with asufficient enzyme concentration in order to obtain the rapid use of thesubstrate in limiting concentration. The difference of OD₃₄₀ observedmakes it possible to deduce the real concentration of this β-ketoestersubstrate of CoA in the reaction.

M.4.1.2. Description of the Enzymatic Test

The catalytic activity of purified MabA was demonstrated byspectrophotometry in the presence of acetoacetyl-CoA and NADPH.

The kinetics of the β-ketoacyl reduction reaction are monitored bymeasuring the absorbance at 340 nm over time, which decreases with theoxidation of the NADPH. The enzymatic reaction is carried out in a fmalvolume of 1 ml (in a quartz cuvette, optical path 1 cm). Thespectrophotometer (UVIKON 923, Bio-Tek Kontron Instruments) is connectedto a thermostatically-controlled bath making it possible to regulate thetemperature of the cuvette at 25° C. A base line is carried out in theabsence of enzyme. The reaction mixture comprises 80 mM of sodiumphosphate buffer, and variable concentrations of NADPH andβ-ketoacyl-CoA. The reaction is triggered by the addition of the enzyme(36 nM to 144 nM). The measurements are carried out over 3 to 5 minutes.

The K_(m) for the NADPH was determined at concentrations of coenzymevarying from 5 to 200 μM and at a fixed concentration (460 μM) ofacetoacetyl-CoA. The K_(m)s for the β-ketoacyl-CoA were determined at afixed concentration, 100 μM, of NADPH. A concentration above 100 μM ledto too much noise at the level of the measurements. It was verified,moreover, that this concentration was saturating.

The K_(m) and V_(max) for the β-ketoacyl-CoAs were measured at thefollowing concentrations: for the acetoacetyl-CoA (C₄), 100-8570 μM; forthe β-ketooctanoyl-CoA (C₈), 4-160 μM; for the β-ketododecanoyl-CoA(C₁₂), 2-32 μM. For the β-ketohexadecanoyl-CoA and the⊕-ketoeicosanoyl-CoA (C₂₀), problems of inhibition by the substrate madeit possible to determine the kinetic parameters and the reactionvelocity was compared at a fixed concentration of 2 μM.

At least two series of experimental points were produced for eachkinetic parameter. The accuracy of these points was verified graphicallyby “double inverse” representation, 1/v=f(1/[S]) (equation (1)). Thekinetic parameters were then determined graphically according to theHanes representation [S]/v=f([S]) (equation (2)) or by calculationaccording to the least error squares method, with GraphPad Prismsoftware Version 2.01. $\begin{matrix}{\frac{1}{V} = {{\frac{K_{m}}{V_{\max}} \cdot \frac{1}{S}} + {\frac{1}{V_{\max}}\quad\left( {{Lineweaver}\text{-}{Burk}} \right)}}} & {{Equation}\quad(1)} \\{\frac{S}{\nu} = {\frac{S}{V\quad\max} + {\frac{Km}{V\quad\max}\quad({Hanes})}}} & {{Equation}\quad(2)}\end{matrix}$

III) Mutagenesis of the Protein MabA and Optimum Purification Methodsfor the Protein MabA, and the Proteins MabA C(60)V, MabA S(144)L andMabA C(60)V/S(144)L

1) Mutagenesis of the Protein MabA

The mutant MabA C(60)V was obtained by site-specific mutagenesis aftercarrying out an inverse PCR. The nucleotide primers were chosen so as tomodify codon 60 of the mabA gene, namely replacement of TGT (cysteine)by GTT (valine). The pET15b::mabA plasmid was used as support for thePCR amplification by DNA polymerase PfuTurbo (Stratagene, USA).

The PCR products were digested with the endonuclease Dpn1 in order toselect the plasmids comprising the mutated gene. The mutated gene wasentirely sequenced in order to verify the absence of secondary mutation.The plasmid carrying the mabA C(60)V gene (pET15b::mabA C(60)V) was thenused to transform the superproducing strain BL21(DE3).

The mutants MabA C(60)V/S(144)L and MabA S(144)L were obtained accordingto the same method as previously.

2) Purification of the Proteins MabA, MabA C(60)V, and MabAC(60)V/S(144)L

Four cultures of 50 ml in LB medium+carbenicillin are carried out. Theturbidity of the medium is measured by spectrophotometry at 600 nm untilthe optical density reaches 0.8 (middle of the exponential growthphase), i.e. after incubation for approximately 4 hours. The expressionof the mabA gene is then induced with 0.8 mM of IPTG for 2 hours at 37°C., then verified by SDS-PAGE.

All of the cultures (4×50 MnCl) are collected by centrifugation (15minutes at 16000 g, at 4° C.) then washed. The pellet obtained is takenup in 4 ml of lysis buffer (see below). Before freezing the suspensionat −80° C. (overnight), a mixture of protease inhibitors (0.113 mg/ml)and lysozyme (0.5 mg/ml) are added to it. The suspension is thawed undergentle stirring at ambient temperature, then treated with DNaseI (5μg/ml) and RNaseA (10 μg/ml) in the presence of 10 mM MgCl₂ for 15minutes at 4° C., under gentle stirring. The whole bacteria and thedebris are eliminated by centrifugation (15 minutes at 3000 g, at 4°C.). A last ultracentrifugation at 44000 g, 15 minutes at −4° C., makesit possible to eliminate any insoluble material. According to the case,before being loaded onto the column, the supernatant (clarified lysate)can be complemented with either 10% (v/v) of glycerol (protein forkinetic studies, or for crystallography of the least stable proteins),or 400 μM NADP⁺ (crystallographic study of the MabA-NADP complex).

Four ml of clarified bacterial lysate (approximately 30 mg of totalproteins) are added to an Ni-NTA agarose column (500 μl, QIAGEN). Thematerial not bound to the resin is recovered by “washings” with bufferwith 5 mM then 50 mM imidazole. The protein MabA is eluted with theelution buffer. When the phosphate buffer is used, the protein isrecovered directly in 50% (v, v) final glycerol, in order to avoidprecipitation. For the crystallography, the protein is concentrated to10-15 mg/ml by ultrafiltration.

Buffers Used:

Proteins for Kinetic Studies:

Lysis buffer: 50 mM potassium phosphate, pH 7.8 containing 500 mM ofNaCl, 5 mM of imidazole

Washing buffers: 50 mM potassium phosphate, pH 7.8 containing 500 mM ofNaCl, 5 and 50 mM of imidazole

Elution buffer: 50 mM potassium phosphate, pH 7.8 containing 500 mM ofNaCl, and 175 mM of imidazole.

Proteins for Crystallography Studies:

Lysis buffer: 50 mM Tris buffer, pH 8.0, supplemented with 300 mM LiSO₄and 5 mM imidazole;

or: 50 mM Tris buffer, pH 8.0, supplemented with 300 mM KCl and 5 mMimidazole.

Washing buffers: 50 mM Tris buffer, pH 8.0, supplemented with 300 mMLiSO₄ and 5 or 50 mM imidazole;

or: 50 mM Tris buffer, pH 8.0, supplemented with 300 mM KCl and 5 or 50mM imidazole.

Elution buffer: 20 mM MES buffer, pH 6.4, 300 mM LiSO₄ and 175-750 mMimidazole;

or: 20 mM PIPES buffer, pH 8.0, supplemented with 300 mM KCl and 175-750mM imidazole.

Note: 1 mM DTT is added to these buffers in the case of the wild-typeprotein.

(MES=2-[N-morpholino]ethane sulphonic acid;PIPES=piperazine-N,N′-bis[2-ethane sulphonic acid])

3) Peptide Sequences of the Proteins Obtained and Nucleotide SequencesCoding for These Proteins

Peptide sequence of the wild-type protein MabA (FabG1) of M.tuberculosis H37Rv in fusion with a poly-His tag (in bold): MGSSHRHHHHSSQLVPRGSH MTATATEGAK PPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSGAPKGLFGVEC DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVINANLTGAFRVA QRASRSMQRN KFGRMTFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARELSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDASYISGAVIPVD GGMGMGH

Peptide sequence of the protein MabA C60V (mutation in bold) in fusionwith a poly-His tag (in bold): MQSSHHHHHH SSGLVPRGSH MTATATEGAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSTARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

Peptide sequence of the protein MabA C60V/S144L (mutations in bold) infusion with a poly-His tag (in bold): MGSSHHHHHH SSGLVPRGSH MTATATEGAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

Nucleotide sequence of the wild-type mabA (fabG1) gene of M.tuberculosis strain H37Rv, in fusion with a sequence coding for apoly-Histidine tag (in capital letters):ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATatgactgccacagccactgaaggggccaaacccccattcgtatcccgttcagtcctggttaccggaggaaaccgggggatcgggctggcgatcgcacagcggctggctgccgacggccacaaggtggccgtcacccaccgtggatccggagcgccaaaggggctgtttggcgtcgaatgtgacgtcaccgacagcgacgccgtcgatcgcgccttcacggcggtagaagagcaccagggtccggtcgaggtgctggtgtccaacgccggcctatccgcggacgcattcctcatgcggatgaccgaggaaaagttcgagaaggtcatcaacgccaacctcaccggggcgttccgggtggctcaacgggcatcgcgcagcatgcagcgcaacaaattcggtcgaatgatattcataggttcggtctccggcagctggggcatcggcaaccaggccaactacgcagcctccaaggccggagtgattggcatggcccgctcgatcgcccgcgagctgtcgaaggcaaacgtgaccgcgaatgtggtggccccgggctacatcgacaccgatatgacccgcgcgctggatgagcggattcagcagggggcgctgcaatttatcccagcgaagcgggtcggcacccccgccgaggtcgccggggtggtcagcttcctggcttccgaggatgcgagctatatctccggtgcggtcatcccggtcgacggcggcatgggtatgggcca c

Nucleotide sequence of the mabA (fabG1) C60V gene (mutated codon inbold) of M. tuberculosis strain H37Rv, in fusion with a sequence codingfor a poly-Histidine tag (in capital letters):ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATatgactgccacagccactgaaggggccaaacccccattcgtatcccgttcagtcctggttaccggaggaaaccgggggatcgggctggcgatcgcacagcggctggctgccgacggccacaaggtggccgtcacccaccgtggatccggagcgccaaaggggctgtttggcgtcgaaGTTgacgtcaccgacagcgacgccgtcgatcgcgccttcacggcggtagaagagcaccagggtccggtcgaggtgctggtgtccaacgccggcctatccgcggacgcattcctcatgcggatgaccgaggaaaagttcgagaaggtcatcaacgccaacctcaccggggcgttccgggtggctcaacgggcatcgcgcagcatgcagcgcaacaaattcggtcgaatgatattcataggttcggtctccggcagctggggcatcggcaaccaggccaactacgcagcctccaaggccggagtgattggcatggcccgctcgatcgcccgcgagctgtcgaaggcaaacgtgaccgcgaatgtggtggccccgggctacatcgacaccgatatgacccgcgcgctggatgagcggattcagcagggggcgctgcaatttatcccagcgaagcgggtcggcacccccgccgaggtcgccggggtggtcagcttcctggcttccgaggatgcgagctatatctccggtgcggtcatcccggtcgacggcggcatgggtatgggcca c

Nucleotide sequence of the mabA (fabG1) C60V/S144L gene (mutated codonsin bold) of M. tuberculosis strain H37Rv, in fusion with a sequencecoding for a poly-Histidine tag (in capital letters):ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATatgactgccacagccactgaaggggccaaacccccattcgtatcccgttcagtcctggttaccggaggaaaccgggggatcgggctggcgatcgcacagcggctggctgccgacggccacaaggtggccgtcacccaccgtggatccggagcgccaaaggggctgtttggcgtcgaaGTTgacgtcaccgacagcgacgccgtcgatcgcgccttcacggcggtagaagagcaccagggtccggtcgaggtgctggtgtccaacgccggcctatccgcggacgcattcctcatgcggatgaccgaggaaaagttcgagaaggtcatcaacgccaacctcaccggggcgttccgggtggctcaacgggcatcgcgcagcatgcagcgcaacaaattcggtcgaatgatattcataggttcggtctccggcCTCtggggcatcggcaaccaggccaactacgcagcctccaaggccggagtgattggcatggcccgctcgatcgcccgcgagctgtcgaaggcaaacgtgaccgcgaatgtggtggccccgggctacatcgacaccgatatgacccgcgcgctggatgagcggattcagcagggggcgctgcaatttatcccagcgaagcgggtcggcacccccgccgaggtcgccggggtggtcagcttcctggcttccgaggatgcgagctatatctccggtgcggtcatcccggtcgacggcggcatgggtatgggcca c

4) Enzymatic Properties

Measurements of the enzymatic kinetics carried out with MabA are thefollowing: acetoacetyl-CoA (C₄:K_(m)=1530±81 μM, k_(cat)=1.9±0.0 s⁻¹),β-ketooctanoyl-CoA (C₈: K_(m)=70±8 μM, k_(cat)=3.5±0.0 s⁻¹),β-ketododecanoyl-CoA (C₁₂: K_(m)=8.3±0.8 μM, k_(cat)=4.3±0.2 s⁻¹).

5) Crystallographical Study

The atomic coordinates of the three-dimensional structure of thecrystals of the protein MabA are represented in FIG. 1, said crystalsmoreover having the following characteristics:

cell parameters:

-   -   a=81.403 angströms, b=116.801 angströms, c=52.324 angströms,

α=β=90.00°, γ=122.30°,

space group: C2,

maximum diffraction=2.05 angströms.

The atomic coordinates of the three-dimensional structure of thecrystals of the protein C(60)V are represented in FIG. 2, said crystalsmoreover having the following characteristics:

cell parameters:

-   -   a=82.230 angströms, b=118.610 angströms, c=53.170 angströms,    -   α=β=90.00°, γ=122.74°,

space group: C2,

maximum diffraction=2.6 angströms.

The atomic coordinates of the three-dimensional structure of thecrystals of the protein C(60)V/S(144)L are represented in FIG. 3, saidcrystals moreover having the following characteristics:

cell parameters:

-   -   a=81.072 angströms, b=117.022 angströms, c=53.170 angströms,    -   α=β=90.00°, γ=122.42°,

space group: C2,

maximum diffraction=1.75 angströms.

BIBLIOGRAPHIC REFERENCES

-   Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang,    Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a    new generation of protein database search programs. Nucleic Acids    Res. 25, 3389-3402.-   Baneijee, A., Dubnau, E., Quemard, A., Balasubramanian, V., Um, K.    S., Wilson, T., Collins, D., de Lisle, G. & Jacobs, W. R. J. (1994).    inhA, a gene encoding a target for isoniazid and ethionamide in    Mycobacteriurh tuberculosis. Science.263, 227-30.-   Barnes, P. F., Bloch, A. B., Davidson, P. T. & Snider, D. E. (1991).    Tuberculosis in patients with immunodeficiency virus infection. N.    Engl. J. Med. 234, 1644-50.-   Basso, L. A., Zheng, R. & Blanchard, J. S. (1996). Kinetics of    inactivation of WT and C243S mutant of Mycobacterium tuberculosis    enoyl reductase by activated isoniazid. J. Am. Chem. Soc. 118,    113014-11302.-   Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N.,    Weissig, H., Shindyalov, I. N. & Boume, P. E. (2000). The protein    data bank. Nucleic Acids Res. 28, 235-242.-   Birge, C. H. & Vagelos, P. R. (1972). Acyl carrier protein.    Purification and properties of β-hydroxyacyl acyl carrier protein    dehydrase. J. Biol. chem. 247, 4930-4938.-   Caughey, I. & Kekwick, R. O. (1982). The Characteristics of Some    Components of the Fatty Acid Synthetase System in the Plastids from    the Mesocarp of Avocado (Persea americana) Fruit. Eur. J. Biochem.    123, 553-561.-   Coakley, W. T., Brown, R. C. & James, C. J. (1973). The Inactivation    of Enzymes by Ultrasonic Cavitation at 20 kHz. Arch. Biochem.    Biophys. 159, 722-729.-   Cohn, D. L., Bustreo, F. & Raviglione, M. C. (1997). Drug-resistant    tuberculosis: review of the worldwide situation and the WHO/IUATLD    global surveillance project. Clinical Infectious Diseases. Clin    Infect Dis 24 (Suppl 1), S121-S130.-   Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C.,    Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E.,    Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T.,    Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S.,    Hamlin, N., Holroyd, S., Homsby, T., Jagels, K. & Barrell, B. G.    (1998). Deciphering the biology of Mycobacterium tuberculosis from    the complete genome sequence. Nature 393, 537-44.-   Constantinides, P. P. & Steim, J. M. (1985). Physical properties of    fatty acyl-CoA. Critical micelle concentrations and micellar size    and shape. J. Biol Chem 260, 7573-7580.-   Dolin, P. J., Raviglione, M. C. & Kochi, A. (1994). Global    tuberculosis incidence and mortality during 1990-2000. Bull World    Health Organ 72, 213-220.-   Gill, S. C. & von Hippel, P. H. (1989). Calculation of protein    extinction coefficients from amino acid sequence data [published    erratum appears in Anal Biochem 1990 September; 189(2):283]. Anal    Biochem 182, 319-26.-   Johnsson, K., King, D. S. & Shultz, P. G. (1995). Studies on the    mechanism of action of isoniazid and ethionarnide in the    chemotherapy of tuberculosis. J. Am. Chem. Soc. 117, 5009-10.-   Kikuchi, S., Takeuchi, T., Yasui, M., Kusaka, T. &    Kolattukudi, P. E. (1989). A very long-chain fatty acid elongation    system in Mycobacterium avium and a possible mode of action of    isoniazid on the system. Agric. Biol. Chem. 53, 1689-98.-   Labesse, G. & Momon, J. (1998). Incremental threading optimization    (TITO) to help alignment and modelling of remote homologues.    Bioinformatics 14, 206-211.-   Labesse, G., Vidal-Cros, A., Chomilier, J., Gaudry, M. & Momon,    J.-P. (1994). Structural comparisons lead to the definition of a new    superfamily of NAD(P)(H)-accepting oxidoreductases: the    single-domain reductases/epimerases/dehydrogenases (the RED family).    Biochem. J. 304, 95-99.-   Laenmili, U. K. (1970). Cleavage of structural proteins during the    assembly of the head of bacteriophage T4. Nature 227, 680-685.-   Luthy, R., Bowie, J. U. & Eisenberg, D. (1992). Assessment of    protein models with three-dimensional profiles. Nature 356, 83-85.-   Persson, B., Kpook, M. & J{circumflex over (0)}rnvall, H. (1991).    Characteristics of short-chain alcohol dehydrogenases and related    enzymes. Eur. J. Biochem. 200, 537-543.-   Phillips, T. A., Van Bogelen, R. A. & Neidhardt, F. C. (1984). Ion    gene product of Escherichia coli is a heat-shock protein. J.    Bacteriol. 159, 283-287.-   Quémard, A., Lacave, C. & Laneelle, G. (1991). Isoniazid inhibition    of mycolic acid synthesis by cell extracts of sensitive and    resistant strains of Mycobacterium aurum. Antimicrob. Agents    Chemother. 35, 1035-9.-   Quemard, A., Mazeres, S., Sut, A., Laneelle, G. & Lacave, C. (1995).    Certain properties of isoniazid inhibition of mycolic acid synthesis    in cell-free systems of M. aurum and M. avium. Biochim Biophys Acta    1254, 98-104.-   Quemard, A., Sacchettini, J. C., Dessen, A., Vilcheze, C., Bittman,    R., Jacobs, W. R. J. & Blanchard, J. S. (1995). Enzymatic    characterization of the target for isoniazid in Mycobacterium    tuberculosis. Biochemistry 34, 8235-41.-   Rawlings, M. & Cronan, J. J. E. (1992). The gene encoding    Escherichia coli acyl carrier protein lies within a cluster of fatty    acid biosynthetic genes. J. Biol. Chem. 267, 5751-5754.-   Rossmann, M. G., Moras, D. & Olsen, K. W. (1974). Chemical and    biological evolution of nucleotide-binding protein. Nature 250,    194-199.-   Rozwarski, D. A., Vilcheze, C., Sugantino, M., Bittman, R. &    Sacchettini, J. C. (1999). Crystal structure of the Mycobacterium    tuberculosis enoyl-ACP reductase, InhA, in complex with NAD+ and a    C16 fatty acyl substrate. J. Biol. Chem. 274, 15582-9.-   Sali, A. & Blundell, T. L. (1993). Comparative protein modelling by    satisfaction of spatial restraints. J. Mol. Biol. 234, 779-815.-   Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular    cloning: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring    Harbor laboratory Press.-   Schulz, H. & Wakil, S. J. (1971). Studies on the mechanism of fatty    acid synthesis. XXV. On the mechanism of beta-ketoacylacyl carrier    protein reductase from Escherichia coli. J. Biol. Chem. 246,    1895-1901.-   Sheldon, P. S., Kekwick, R. G. O., Sidebottom, C., Smith, C. G. &    Slabas, A. R. (1990). 3-Oxoacyl-(acyl-carrier protein) reductase    from avocado (Persea americana) fruit mesocarp. Biochem. J. 271,    713-720.-   Sheldon, P. S., Kekwick, R. G. O., Smith, C. G., Sidebottom, C. &    Slabas, A. R. (1992). 3-Oxoacyl-[ACP] reductase from oilseed rape    (Brassica napus). Biochim. Biophys. Acta. 1120, 151-159.-   Shen, Z. & Byers, D. M. (1996). Isolation of Vibrio harveyi acyl    carrier protein and the fabG, acpP, and fabF genes involved in fatty    acid biosynthesis. J. Bacteriol. 178, 571-573.-   Shimakata, T. & Stumpf, P. K. (1982). Purification and    Characterizations of β-Ketoacyl-[Acyl-Carrier-Protein] Reductase,    β-Hydroxyacyl-[Acyl-Carrier-Protein] Dehydrase, and    Enoyl-[Acyl-Carrier-Protein] Reductase from Spinacea oleracea    Leaves. Arch. Biochem. Biophys. 218, 77-91.-   Studier, F. W. & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA    polymerase to direct selective high-level cloned genes. J. Mol.    Biol. 189, 113-130.-   Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W.    (1990). Use of T7 RNA polymerase to direct expression of cloned    genes. Meth. Enzymol. 185, 60-89.-   Takayama, K., Wang, L. & David, H. L. (1972). Effect of isoniazid on    the in vivo mycolic acid synthesis, cell growth, and viability of    Mycobacterium tuberculosis. Antimicrob Agents Chemother 2, 29-35.-   Weeks, G. & Wakil, S. J. (1968). Studies on the mechanism of fatty    acid synthesis 18. Preparation and general properties of the enoyl    acyl carrier protein reductases from Escherichia coli. J. Biol.    Chem. 243, 1180-1189.-   Winder, F. G. & Collins, P. B. (1970). Inhibition by isoniazid of    synthesis of mycolic acids in Mycobacterium tuberculosis. J. Gen.    Microbiol. 63, 41-48.

1-40. (canceled)
 41. Protein MabA, alsocalled protein FabG1, recombinantin purified form, or recombinant proteins derived from the protein: MabAby mutation of one or more amino acids, said derived proteins being inpurified form, and having a NADPH-dependent β-ketoacyl reductaseactivity.
 42. Purified recombinant protein MabA according to claim 41,said protein being a protein of mycobacteria.
 43. A method for producingthe recombinant protein MabA or derived recombinant proteins in purifiedform according to claim 42, as by transforming strains of E. coli with aplasmid containing a sequence comprising the gene coding for the proteinMabA, or comprising a sequence coding for a protein derived from MabA,followed by a purification stage during which: the abovementionedrecombinant E. coli bacteria are washed in a washing buffer, then takenup in a lysis buffer, and lysed by a freeze/thaw cycle in the presenceof protease inhibitors and lysozyme, after treatment by DNAse I andRNAse A, in the presence of MgCl₂, and centrifugation, the lysissupernatant of the bacteria obtained in the preceding stage, to which10% (v/v) of glycerol, or 400 μM of NADP⁺ is added, is applied to anNi-NTA agarose resin column, after several washings with 5 mM bufferthen 50 mM imidazole, the protein MabA, or the derived protein, iseluted with the elution buffer.
 44. The process according to claim 43, arecombinant protein MabA or derived recombinant proteins in purifiedform wherein the different bacteria washing, lysis, washing, and elutionbuffers, are the following: bacteria washing buffer: 10 mM potassiumphosphate, pH 7.8, lysis buffer: 50 mM potassium phosphate, pH 7.8containing 500 mM of NaCl, 5 mM of imidazole, washing buffer: 50 mMpotassium phosphate, pH 7.8 containing 500 mM of NaCl, 5 and 50 mM ofimidazole, elution buffer: 50 mM potassium phosphate, pH 7.8 containing500 mM of NaCl, and 175 mM of imidazole.
 45. The method according toclaim 43, wherein the different bacteria washing, lysis, washing, andelution buffers, are the following: bacteria washing buffer: Tris 10 mM,pH 8.0, lysis buffer: 50 mM Tris buffer, pH 8.0, supplemented with 300mM LiSO₄ and 5 mM imidazole; or 50 mM Tris buffer, pH 8.0, supplementedwith 300 mM KCl and 5 mM imidazole, washing buffer: 50 mM Tris buffer,pH 8.0, supplemented with 300 mM LiSO₄ and 5 or 50 mM imidazole, or 50mM Tris buffer, pH 8.0, supplemented with 300 mM KCl and 5 or 50 mMimidazole. elution buffer: 20 mM MES buffer, pH 6.4, LiSO4 300 mM and175-750 mM imidazole; or 20 mM PIPES buffer, pH 8.0, supplemented with300 mM KCl and 175-750 mM imidazole, 1 mM DTT being added to thesebuffers in the case of the wild-type protein MabA.
 46. Proteins derivedfrom the protein MabA according to claim 41, characterized in that theycorrespond to the protein MabA the amino acid sequence SEQ ID NO: 1 ofwhich is the following: MTATATEGAK PPFVSRSVLV TGGNRGIGLA IAQRLAADGHKVAVTHRGSG APKGLFGVEC DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRMTEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGSWGIGNQ ANYAASKAGVIGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGVVSFLASEDAS YISGAVIPVD GGMGMGH

in which the cysteine in position 60 is replaced by a valine residue,and/or the glycine in position 139 is replaced by an alanine or aserine, and/or the serine in position 144 is replaced by a leucineresidue.
 47. Protein derived from the protein MabA according to claim41, characterized in that it corresponds to the protein MabA in whichthe cysteine in position 60 is replaced by a valine residue, saidderived protein, also called C(60)V, corresponding to the followingsequence SEQ ID NO 3: MTATATEGAK PPFVSRSVLV TGGNRGIGLA IAQRLAADGHKVAVTHRGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRMTEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGSWGIGNQ ANYAASKAGVIGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGVVSFLASEDAS YISGAVIPVD GGMGMGH


48. Protein derived from the protein MabA according to claim 41,characterized in that it corresponds to the protein MabA in which theserine in position 144 is replaced by a leucine residue, said derivedprotein, also called S(144)L, corresponding to the following sequenceSEQ ID NO 5: MTATATEGAK PPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSGAPKGLFGVEC DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVINANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARELSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDASYISGAVIPVD GGMGMGH


49. Protein derived from the protein MabA according to claim 41,characterized in that it corresponds to the protein MabA in which thecysteine in position 60 is replaced by a valine residue, and the serinein position 144 is replaced by a leucine residue, said derived protein,also called C (60)V/S(144)L, corresponding to the following sequence SEQID NO 7: MTATATEGAK PPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSGAPKGLFGVEV DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVINANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARELSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDASYISGAVIPVD GGMGMGH


50. Protein derived from the protein MabA according to claim 41,characterized in that it corresponds to the protein MabA in which thecysteine in position 60 is replaced by a valine residue, the glycine inposition 139 is replaced by an alanine or a serine, and the'serine inposition 144 is replaced by a leucine residue, said derived protein,also called C(60)V/G(139) [A or S]/S(144)L, corresponding to thefollowing sequence SEQ ID NO 8: MTATATEGAK PPFVSRSVLV TGGNRGIGLAIAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQG PVEVLVSNAGLSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIXS VSGLWGIGNQANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKRVGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X represents A or S.
 51. Protein MabA corresponding to thesequence SEQ ID NO: 1, or proteins derived from the protein MabAaccording to claim 6 or proteins corresponding to the sequences SEQ IDNO: 3, 5, 7, or 8, characterized in that they are modified so that theyinclude one or more mutations making it possible to change thespecificity of the protein NADPH to NADH.
 52. Modified MabA proteinsaccording to claim 51, corresponding to the following sequences: thesequence SEQ ID NO: 9, corresponding to the sequence SEQ ID NO: 1comprising the mutations N24D(or E), and/or H46D, namely the followingsequence: MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RCSGAPKGLFGVEC DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVINANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARELSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDASYISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the sequenceSEQ ID NO: 10, corresponding to the sequence SEQ ID NO: 3 comprising themutations N24D(or E), and/or H46D, namely the following sequence:MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEVDVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVAQRASRSMQRN KFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANVVAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVDGGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the sequenceSEQ ID NO: 11, corresponding to the sequence SEQ ID NO: 5 comprising themutations N24D(or E), and/or H46D, namely the following sequence:MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVECDVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVAQRASRSMQRN KFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANVVAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVDGGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the sequenceSEQ ID NO: 12, corresponding to the sequence SEQ ID NO: 7 comprising themutations N24D(or E), and/or H46D, namely the following sequence:MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEVDVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVAQRASRSMQRN KFGRMIFIGS VSGLWCIGNQ ANYAASKAGV IGMARSIARE LSKANVTANVVAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVDGGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the sequenceSEQ ID NO: 13, corresponding to the sequence SEQ ID NO: 8 comprising themutations N24D(or E), and/or H46D, namely the following sequence:MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEVDVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVAQRASRSMQRN KFGRMIFIXS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANVVAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVDGGMGMGH

in which X₁ represents D or E, and X₂ represents H or D.
 53. ProteinMabA corresponding to the sequence SEQ ID NO: 1, or proteins derivedfrom the protein MabA according to claim 46, characterized in that theyare modified by insertion, on the N-terminal side, of a poly-histidinetag such as the following sequence SEQ ID NO: 14: MGSSHHHHHH SSGLVPRGSH.54. Modified proteins MabA according to claim 53, corresponding to thefollowing sequences: the sequence SEQ ID NO: 15, corresponding to thecombination of the sequence SEQ ID NO: 14 and the sequence SEQ ID NO: 1,namely the following sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the sequence SEQ ID NO: 16, corresponding to the combination of thesequence SEQ ID NO: 14 and the sequence SEQ ID NO: 3, namely thefollowing sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLVTGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFCVEV DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the sequence SEQ ID NO: 17, corresponding to the combination of thesequence SEQ ID NO: 14 and the sequence SEQ ID NO: 5, namely thefollowing sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLVTGGNRGIGLA IAQRLAADGH KVAVTRRGSG APKGLFGVEC DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the sequence SEQ ID NO: 18, corresponding to the combination of thesequence SEQ ID NO: 14 and the sequence SEQ ID NO: 7, namely thefollowing sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLVTGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the sequence SEQ ID NO: 19, corresponding to the combination of thesequence SEQ ID NO: 14 and the sequence SEQ ID NO: 9, namely thefollowing sequence: MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLVTGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEC DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the sequenceSEQ ID NO: 20, corresponding to the combination of the sequence SEQ IDNO: 14 and the sequence SEQ ID NO: 10, namely the following sequence:MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGHKVAVTX₂RGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRMTEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGSWGIGNQ ANYAASKAGVIGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGVVSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the sequenceSEQ ID NO: 21, corresponding to the combination of the sequence SEQ IDNO: 14 and the sequence SEQ ID NO: 11, namely the following sequence:MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGHKVAVTX₂RGSG APKGLFGVEC DVTDSDAVDR AFTAVEERQG PVEVLVSNAG LSADAFLMRMTEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGLWGIGNQ ANYAASKAGVIGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGVVSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the sequenceSEQ ID NO: 22, corresponding to the combination of the sequence SEQ IDNO: 14 and the sequence SEQ ID NO: 12, namely the following sequence:MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGHKVAVTX₂RGSC APKGLFGVEV DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRMTEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGLWGIGNQ ANYAASKAGVIGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGVVSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the sequenceSEQ ID NO: 23, corresponding to the combination of the sequence SEQ IDNO: 14 and the sequence SEQ ID NO: 13, namely the following sequence:MGSSHHHHHH SSGLVPRGSH MTATATEGAK PPFVSRSVLV TGGX₁RGIGLA IAQRLAADGHKVAVTX₂RGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRMTEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIXS VSGLWGIGNQ ANYAASKAGVIGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGVVSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D.
 55. ProteinMabA corresponding to the sequence SEQ ID NO: 1, or proteins derivedfrom the protein MabA according to claim 46, having an N-terminal GSHsequence, namely the following sequences: the following sequence SEQ IDNO: 24, corresponding to the combination of the GSH sequence and thesequence SEQ ID NO: 1, GSH MTATATEGAK PPFVSRSVLV TGGNRGIGLA IAQRLAADGHKVAVTHRGSG APKGLFGVEC DVTDSDAVDR AFTAVEEHQG PVEVLVSNAG LSADAFLMRMTEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGSWGIGNQ ANYAASKAGVIGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKR VGTPAEVAGVVSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 25, corresponding to the combinationof the GSH sequence and the sequence SEQ ID NO: 3, GSH MTATATEGAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 26, corresponding to the combinationof the GSH sequence and the sequence SEQ ID NO: 5, GSH MTATATEGAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 27, corresponding to the combinationof the GSH sequence and the sequence SEQ ID NO: 7, GSH MTATATEGAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 28, corresponding to the combinationof the GSH sequence and the sequence SEQ ID NO: 9, GSH MTATATEGAKPPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the followingsequence SEQ ID NO: 29, corresponding to the combination of the GSHsequence and the sequence SEQ ID NO: 10, GSH MTATATEGAK PPFVSRSVLVTGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the followingsequence SEQ ID NO: 30, corresponding to the combination of the GSHsequence and the sequence SEQ ID NO: 11, GSH MTATATEGAK PPFVSRSVLVTGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEC DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the followingsequence SEQ ID NO: 31, corresponding to the combination of the GSHsequence and the sequence SEQ ID NO: 12, GSH MTATATEGAK PPFVSRSVLVTGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGSVSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the followingsequence SEQ ID NO: 32, corresponding to the combination of the GSHsequence and the sequence SEQ ID NO: 13, GSH MTATATEGAK PPFVSRSVLVTGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQGPVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIXSVSCLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQGALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D.
 56. ProteinMabA corresponding to the sequence SEQ ID NO: 1, or proteins derivedfrom the protein MabA according to claim 46, in which the first sevenamino acids are deleted, namely the following sequences: the followingsequence SEQ ID NO: 33, corresponding to the sequence SEQ ID NO: 1 thefirst seven amino acids of which are deleted: GAK PPFVSRSVLV TGGNRGIGLAIAQRLAADGH KVAVTHRGSG APKGLFGVEC DVTDSDAVDR AFTAVEEHQG PVEVLVSNAGLSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGSWGIGNQANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKRVGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 34, corresponding to the sequence SEQID NO: 3 the first seven amino acids of which are deleted: GAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 35, corresponding to the sequence SEQID NO: 5 the first seven amino acids of which are deleted: GAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 36, corresponding to the sequence SEQID NO: 7 the first seven amino acids of which are deleted: GAKPPFVSRSVLV TGGNRGIGLA IAQRLAADGH KVAVTHRGSG APKGLFGVEV DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGLWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

the following sequence SEQ ID NO: 37, corresponding to the sequence SEQID NO: 9 the first seven amino acids of which are deleted: GAKPPFVSRSVLV TGGX₁RGIGLA IAQRLAADGH KVAVTX₂RGSG APKGLFGVEC DVTDSDAVDRAFTAVEEHQG PVEVLVSNAG LSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRNKFGRMIFIGS VSGSWGIGNQ ANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDMTRALDERIQQ GALQFIPAKR VGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the followingsequence SEQ ID NO: 38, corresponding to the sequence SEQ ID NO: 10 thefirst seven amino acids of which are deleted: GAK PPFVSRSVLV TGGX₁RGIGLAIAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQG PVEVLVSNAGLSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGSWGIGNQANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKRVGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the followingsequence SEQ ID NO: 39, corresponding to the sequence SEQ ID NO: 11 thefirst seven amino acids of which are deleted: OAK PPFVSRSVLV TGGX₁RGIGLAIAQRLAADGH KVAVTX₂RGSG APKGLFGVEC DVTDSDAVDR AFTAVEEHQG PVEVLVSNAGLSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMTFIGS VSGLWGIGNQANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKRVGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the followingsequence SEQ ID NO: 40, corresponding to the sequence SEQ ID NO: 12 thefirst seven amino acids of which are deleted: GAK PPFVSRSVLV TGGX₁RGIGLAIAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQG PVEVLVSNAGLSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIGS VSGLWGIGNQANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKRVGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D, the followingsequence SEQ ID NO: 41, corresponding to the sequence SEQ ID NO: 13 thefirst seven amino acids of which are deleted: GAK PPFVSRSVLV TGGX₁RGIGLAIAQRLAADGH KVAVTX₂RGSG APKGLFGVEV DVTDSDAVDR AFTAVEEHQG PVEVLVSNAGLSADAFLMRM TEEKFEKVIN ANLTGAFRVA QRASRSMQRN KFGRMIFIXS VSGLWGIGNQANYAASKAGV IGMARSIARE LSKANVTANV VAPGYIDTDM TRALDERIQQ GALQFIPAKRVGTPAEVAGV VSFLASEDAS YISGAVIPVD GGMGMGH

in which X₁ represents D or E, and X₂ represents H or D.
 57. Proteinsaccording to claim 41, characterized by having specific enzymaticactivity of the substrates of the long-chain type β-ketoacyl. 58.Proteins according to claim 41, the main characteristics of thethree-dimensional structure of which, at a resolution of 1.6-2.0angstroms, detected by X-ray diffraction analysis of the crystals ofsaid proteins, are as represented in FIG. 1 for the recombinant proteinMabA corresponding to the sequence SEQ ID NO: 15, in FIG. 2 for thederived protein MabA C(60)V corresponding to the sequence SEQ ID NO: 16,and in FIG. 3 for the derived protein MabA C(60)V/S(144)L correspondingto the sequence SEQ ID NO:
 17. 59. Proteins according to claim 41, incrystallized form.
 60. Crystals of proteins according to claim 59, asobtained by the hanging-drop vapour diffusion method, by mixing saidproteins (1 μl of a 10 mg/ml solution) with a solution of polyethyleneglycol, CsCl (150-300 mM), and glycerol (10%) in a buffer (PIPES) at pH6.2.
 61. Crystals of proteins according to claim 59, as obtainedaccording to the crystallization method described in claim 20, saidmethod being carried out from purified proteins.
 62. Crystals of therecombinant protein MabA corresponding to the sequence SEQ ID NO: 15according to claim 59, the atomic coordinates of the three-dimensionalstructure of which are represented in FIG. 1, and having the followingcharacteristics: cell parameters: a=81.403 angströms, b=116.801angströms, c=52.324 angströms, α=β=90.00°, γ=122.30°, space group: C2,maximum diffraction=2.05 angströms.
 63. Crystals of the protein C(60) Vcorresponding to the sequence SEQ ID NO: 16, according to claim 59, theatomic coordinates of the three-dimensional structure of which arerepresented in FIG. 2, and having the following characteristics: cellparameters: a=82.230 angströms, b=118.610 angströms, c=53.170 angströms,α=β=90.00°, γ=122.74°, space group: C2, maximum diffraction=2.6angströms.
 64. Crystals of the protein C(60)V/S(144) L corresponding tothe sequence SEQ ID NO: 18 according to claim 59, the atomic coordinatesof the three-dimensional structure of which are represented in FIG. 3,and having the following characteristics: cell parameters: a=81.072angströms, b=117.022 angströms, c=53.170 angströms, α=β=90.00°,γ=122.42°, space group: C2, maximum diffraction=1.75 angströms. 65.Crystals of proteins according to claim 57, in which said proteins arebound to a ligand, namely a molecule capable of binding to the proteinMabA.
 66. Nucleotide sequence coding for a protein derived from theprotein MabA as defined in claim
 41. 67. Recombinant nucleotide sequencecomprising the nucleotide sequence coding for the protein MabA, orcomprising a nucleotide sequence coding for a protein derived from theprotein MabA according to claim 41, in combination with the elementsnecessary for the transcription of this sequence, in particular with atranscription promoter and terminator.
 68. Vector, in particularplasmid, containing a nucleotide sequence according to claim
 67. 69.Host cells transformed by a vector according to claim 68, said cellsbeing chosen from the bacteria, or any other microorganism used for theproduction of proteins.
 70. Process for the preparation of therecombinant protein MabA in purified form, or of recombinant proteinsderived from the protein MabA according to claim 41, characterized inthat said process comprises the following stages: transforming of cellsusing a recombinant vector, culturing of the cells thus transformed, andrecovery of said proteins produced by said cells, and purifying saidproteins.
 71. A method for screening ligands of the protein MabA,comprising the following stages: being brought into the presence of therecombinant protein MabA in purified form, or a recombinant proteinderived from the protein MabA according to claim 41, detection of anybond between said protein and the ligand tested by measurement, afterfluorescence excitation, in particular at 300 nm, of the intensity offluorescence of said protein emitted between 300 and 400 nm(corresponding essentially to the emission of fluorescence of the singletryptophan W145), and comparison of the intensity of fluorescenceemitted in a test in the absence of ligand, the binding of a ligand inthe MabA active site being characterized by a quenching of fluorescence.72. A method for screening ligands inhibiting the protein MabA,comprising the following stages: being brought into the presence of therecombinant protein MabA in purified form, or a recombinant proteinderived from the protein MabA according to claim 41, in a reactionmedium comprising a substrate, the coenzyme NADPH and the ligand tested,detection of a potential inhibiting ability of the ligand tested, bymeasurement of the enzymatic activity of said protein by kineticmeasurement of the absorbance, in particular at 340 nm, and comparisonof the gradient of the optical density curve as a function of time withthe gradient obtained in a test in the absence of ligand.
 73. A methodfor screening ligands of-the protein MabA, comprising the followingstages: being brought into the presence of the recombinant protein MabAin purified form, or of a recombinant protein derived from the proteinMabA according to claim 41, with the ligand tested, analysis of thethree-dimensional structure of the complex formed in soluble phasebetween said protein and said ligand, in particular by NMR, and byfluorescence.
 74. Method for screening ligands of the protein MabA,characterized in that said method comprises the following stages:co-crystallization of the ligand tested and the recombinant protein MabAin purified form, or of a recombinant protein derived from the proteinMabA according to claim 41, or soaking of the crystals of the proteinMabA or of a derived recombinant protein, in optimized solutionscontaining potential ligands, analysis of the three-dimensionalstructure of the abovementioned crystals, in particular by X-raydiffraction (with a view to selecting the ligands having an optimumability to occupy and block the active site of said proteins).
 75. Amethod for designing or screening ligands of the protein MabA,comprising analyzing the coordinates of the three-dimensional structureof the recombinant protein MabA in purified form, or a recombinantprotein derived from the protein MabA according to claim 41, saidcoordinates being represented in FIGS. 1 to 3, if appropriate incombination with the coordinates of the active site of these proteins.76. Method for designing or screening, the protein MabA, or proteinswith a structure close to the protein MabA, comprising the analyzing thecoordinates of a three-dimensional structure of the recombinant proteinMabA in purified form, or of a recombinant protein derived from theprotein MabA according to claim 41, said coordinates being representedin FIGS. 1 to 3, for screening in silico of the virtual combinatoriallibraries of potential ligands, and the detection and rationalstructural optimization of the molecules capable of binding to saidprotein.
 77. Method of rational design of ligands of the protein MabA,said method being carried out starting with known inhibitors of MabA orinhibitors of proteins homologous to MabA, for which the finethree-dimensional structure of the complex between said inhibitor andthe recombinant protein MabA in purified form, or a recombinant proteinderived from the protein MabA according to claim 41, was determined, andrational structural optimization of said inhibitors.